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Review Paper
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.
SUMMARY
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 ÒfieldÓ to the final maintenance of
thecompound eye. The researchers
usedDNA microarray technology to get an overview of the Ògenetic cascadeÓ 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 Òmaster controlÓ gene and the underlying cause of
eyemorphogenesis. For
greateraccuracy, two different Drosophila full-genome oligonucleotide
microarraysÑroDROMEGaand DrosGenomeÑwere 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.
CRITIQUE
Figure 1A:
Figure1A
is a Venn diagram comparing the two probe arraysÕ 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.
Figure 1B:
Figure1B
classifies the 254 eyeless-induced genes for which a molecularfunction
could be determined. It showsboth
the diversity of the activated genesÕ functionsÑranging from receptors to
kinasesto cytoskeletal elementsÑand the prevalence of transcription factors and
signaltransducers. It is obvious
fromthis figure that eyeless has a significant impact on development.
Table 1:
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 2:
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.
Figure 2:
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
researcherÕs 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.
CONCLUSIONS
Overall,the
figures and tables presented in this paper support the researchersÕ 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
microarraysÕdetecting abilities.
Though thesemay not be necessary to prove the researchersÕ 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 proteinsÕ 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.