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Analysis of the EyeDevelopmental Pathway in Drosophila Using DNA Microarrays.
Lydia Michaut, SusanneFlister, Martin Neeb, Kevin P. White, Ulrich Certa, and Walter J. Gehring
Proceedings of the National Academy of Sciences of the UnitedStates of America, Vol. 100, No. 7, 2003 April 1, 4024-4029.
Previous studiesof ectopic eye development in Drosophila flies proved that eyeformation could be induced by targeted expression of the eyeless gene aswell as homologous genes from other organisms. Because many eye developmental genes are so highly conservedthat a gene from a squid can cause normal fly eye formation, scientists havebeen working to understand the developmental pathway. The methods described in this paper canbe used to better understand the genetic basis of eye development, from theinitial establishment of an eye “field” to the final maintenance of thecompound eye. The researchers usedDNA microarray technology to get an overview of the “genetic cascade” by analyzingthe expression of various genes involved in ectopic eye development. Most of these genes are transcriptionfactors activated early in retinal differentiation by eyeless, which isbelieved to be the “master control” gene and the underlying cause of eyemorphogenesis. For greateraccuracy, two different Drosophila full-genome oligonucleotide microarrays—roDROMEGaand DrosGenome—were used to compare gene expression in both wild-type leg discsand leg discs in which eyeless was being ectopicallyexpressed. Because eyeless can act asa transcription factor for other processes in other tissues, the researchersthen analyzed the endogenous expression of selected genes from eye discs todetermine which genes were eye-specific. They found 371 genes that are expressed in the eye discs andup-regulated when an eye is ectopically induced in leg discs. Many of these genes encodetranscription factors involved in developmental processes. Some of the identified genes werealready known to act downstream of eyeless, while other genes had notyet been associated with eye development.
Figure1A is a Venn diagram comparing the two probe arrays’ detection of the 371 eyeless-inducedgenes. 228 genes were detected by theroDROMEGa array alone, compared to 198 genes detected by the DrosGenome arrayalone. 55 genes were detected byboth arrays and are listed in further detail in Table 1.
Thisdiagram effectively demonstrates why two microarrays are needed: their accuracydepends on the selection of the oligonucleotide sequences chosen to representeach gene. The use of twodifferent arrays reduces the number of false positives and allows betteranalysis of gene expression.
Figure1B classifies the 254 eyeless-induced genes for which a molecularfunction could be determined. It showsboth the diversity of the activated genes’ functions—ranging from receptors to kinasesto cytoskeletal elements—and the prevalence of transcription factors and signaltransducers. It is obvious fromthis figure that eyeless has a significant impact on development.
Table1 lists the 55 genes detected by both microarrays; these genes are found both inthe eye discs and during ectopic eye formation. The table also lists the fold induction for each gene;however, the two arrays detected different fold inductions for most of thegenes, reinforcing the need for more than one array and making it more difficultto determine the amount of transcription of each gene.
Thistable also provides evidence that some genes not previously thought to beassociated with eye formation are actually involved in the process. For example, the chit gene, whichis synthesized in body fat and encodes a chitinase-like disc growth factor, istranscribed in both leg and eye discs, especially during eptopic eyeformation. These results indicatethat chit has autonomous role in disc development and eye differentiation. Other known genes, such as the actinbundle assembly protein quail, the transcription factors fru and ken, and theGTPase rac2, are significantly induced during eptopic eye development,pointing to some previously unnoticed role in the process.
Table 2lists genes discussed in the text but not present in Table 1, and it includesthe DNA microarray values for each gene. Again, by providing evidence of their up-regulation during eptopic eyeformation, this table suggests that some genes involved in other developmentalprocesses, for example the transcription factor net and thesignal transducers sprint and Sur-8, also haveroles in eye development.
Figure2 presents the only pictorial evidence in this paper, showing endogenousexpression of various eyeless-induced genes in wild-type eyediscs. Panel 2A shows detection ofthe quail protein using a 6B9 monoclonal antibody; the protein is visiblein the eye disc posterior to the morphogenetic furrow. Panels 2B through 2N show in situ hybridizationusing digoxigenin-labeled antisense RNA probes (they controlled signalspecificity with sense RNA probes, which were not shown) corresponding to thegene in each panel. The knowngenes shown include Sur-8, ken, and sprint; these areall visible in rows of cells posterior to the morphogenetic furrow. Several uncharacterized genes are alsoshown and are visible around the morphogenetic furrow, although some are morevisible than others.
Whencombined with the data from Tables 1 and 2, Figure 2 provides even more supportfor the researcher’s proposal that these genes are involved in eyeformation. Not only are theypresent during eptopic growth in the leg discs; they are also visibly endogenouslyexpressed in the eye discs, performing a variety of functions.
Overall,the figures and tables presented in this paper support the researchers’ theoryabout the involvement of multiple proteins in the eye development pathway. However, several tables referenced inthe text are not published with the paper, but rather as supporting informationon the PNAS webpage. Table 3, forinstance, details the 38 transcription factors found in the eye discs and in eptopiceye formation, and Table 7 provides more information about the two microarrays’detecting abilities. Though thesemay not be necessary to prove the researchers’ point about the potentialdevelopmental pathway, it is always helpful to be able to see the evidencebeing cited, especially when it concerns the accuracy of the microassays.
Also,the theory itself is vague. Itprovides a general overview of the developmental pathway, detailing specificgenes and suggesting possible roles of their encoded proteins, but there are notests of function to support these ideas. Granted, the functions of several of these proteins are already known,but it would be interesting to see what happens to eye development when mutatedcopies of these gene are present. Tests of function for the unknown genes in particular would be of interest;the researchers hypothesized about the proteins’ potential functions but didnot explore the genes beyond their expression patterns and probable structures.
Thedata presented is sufficient to warrant further research on this topic, and itprovides 371 possible genes with which an experiment may be begun. Since several of the genes, known or previouslyunknown, may have homologs in other species, it might be well worth aninvestigation into their specific involvement in eye development. It might also be interesting to findout if a third or fourth microassay produces the same 371 genes, or if thereare still more waiting to be discovered. Further research might ultimately allow comparisons between thedevelopment of the compound insect eye and the development of the camera-typemammal eye, since so many of the proteins involved, like eyeless, are highly conserved.