Profiling Patterned Transcripts in <em>Drosophila</em> Embryos

Abstract

Pattern formation represents an initial event in the development of multicellular organisms. Subsequent organismal complexity is generated from translation of the early embryonic pattern into differentiated cell types and tissues. Our current understanding of the molecular mechanisms of patterning in Drosophila embryogenesis derives in large part from an analysis of genes corresponding to embryonic lethal mutants that display defects in larval cuticular morphology (Jurgens et al. 1984; Nusslein-Volhard et al. 1984; Wieschaus et al. 1984; Schupbach and Wieschaus 1989). Molecular characterization of genes essential for embryonic patterning has revealed that these genes code almost exclusively for components of signal transduction cascades and their associated transcription factors. Taken together, molecular and genetic studies have furnished us with a comprehensive understanding of how patterned transcription can arise in Drosophila embryogenesis. From a developmental perspective, the importance of patterned transcription is clear: Placement of gene products in a subset of embryonic cells causes these cells to assume fates different from their nonexpressing neighbors.

In contrast to our comprehensive understanding of how embryonic patterns are established in Drosophila, our understanding of the molecular mechanisms that are employed in translating pattern into differentiated tissues and cell types is more limited. It is notable that many of the transcriptionally regulated targets of the pattern-establishing signaling cascades have escaped detection using standard methods of genetic surveillance. It has been suggested that patterned expression of single genes belonging to subgroups that are expressed in overlapping domains may promote establishment of the final differentiated state without functioning as an absolute determinant of differentiation (Wieschaus 1996). Effects of mutation in genes such as these are expected to be subtle or transient.

As a complement to classical genetic screens, reverse genetic approaches have been exploited to identify a broader assortment of developmentally important loci in Drosophila. By definition, the output of a reverse genetic screen is not limited by phenotype(s). More importantly, the demonstration that gene expression patterns often presage an essential function for the corresponding gene product within the spatially restricted domain of gene expression validates the use of reverse genetics for the identification of developmentally regulated gene products. Genes essential for both pattern establishment and differentiation might be spatially and/or temporally regulated and therefore will be recovered in expression screens.

Enhancer detection screens have traditionally represented the reverse genetic method of choice in Drosophila (O'Kane and Gehring 1987; Bellen et al. 1989; Bier et al. 1989; Torok et al. 1993). The large collections of P-element enhancer detection insertions that are now widely available have had a tremendous impact on all aspects of Drosophila biology. In particular, P-element enhancer detection lines have facilitated identification and characterization of numerous developmentally regulated genes and cell-specific markers in Drosophila. There are, however, some caveats associated with enhancer detection as a reverse genetic screening method. These studies require a substantial investment of personnel and resources in the generation of founder fly lines, the identification of P-element enhancer detection carriers, and the maintenance of fly lines. In addition, gene identification in P-element enhancer detection lines can be difficult and oftentimes impossible because many P-element enhancer detection fly lines harbor multiple P-element insertions. Finally, anecdotal evidence suggests that expression of the reporter gene accurately reflects the expression of a nearby gene in as few as 50% of the enhancer detection lines.

An alternative approach for identifying genes with spatially or temporally regulated expression during embryonic development is the analysis of mRNA expression patterns in situ using randomly isolated cDNAs as hybridization probes (Gawantka et al. 1998; Kopczynski et al. 1998; Liang and Biggin 1998; Kudoh et al. 2001). This approach has three important advantages over traditional enhancer detection screens: (1) Reagent stocks are cDNAs, thus eliminating the labor and cost associated with maintaining thousands of fly lines; (2) probes correspond to individual cDNAs, thus leading to immediate gene identification; and (3) expression patterns correspond to endogenous mRNAs, thus establishing physiological relevance. Until now, fundamental difficulties associated with the availability and processing of large numbers of sequence-verified cDNAs have limited the utility of expression studies such as these. We have employed our own sequencing studies and the Drosophila genome project (Adams et al. 2000) as an immediate solution to the first of these problems and a robotic screening method to provide a solution to the second.

In this paper, we describe a high-throughput screen for the identification of spatially restricted transcripts. Exploiting robotic automation, we analyzed 778 sequence-selected genes from a 0- to 4-h embryonic cDNA library by hybridization in situ. As expected, we identified several previously characterized genes with documented roles in patterning of the early Drosophila embryo. More exciting was our discovery that many novel and uncharacterized genes exhibit spatially restricted patterns of expression in developing embryos, suggestive of their special roles as determinants and/or markers of tissue-specific fates. Taken together, our studies represent a first step in an important phase in translational genomic studies—positioning new gene products in already characterized embryonic patterning pathways.

cDNA gene ontologies underrepresented in comparison to the genome are darkly shaded; overrepresented cDNA gene ontologies are lightly shaded.

Acknowledgments

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