REVIEW of the PAPER
ANALYSIS OF THE EYE DEVELOPMENTAL PATHWAY IN DROSOPHILA USING DNA MICROARRAYS
Lydia Michaut, Susanne Flister, Martin Neeb, Kevin P. White, Ulrich Certa, and Walter J. Gehring
Proceedings of the National Academy of Sciences, Volume 100, Number 7, April 1, 2003, pages 4024-4029.
REVIEW:
Introduction:
Pax-6 genes are known transcription factors involved in the development of the eye. It has been shown that these genes are evolutionarily conserved in vertebrates and invertebrates. They encode for proteins that initiate a cascade all the other proteins that are necessary for eye formation. eyeless (ey) is one such Pax-6 gene that has been identified in Drosophila. The expression of this gene initiates the cascade that controls the development of the compound eye found in Drosophila.
Previous studies have shown that expression of ey early in development is sufficient for complete development of ectopic eyes in the wings, antennae, and legs. It has also been shown that the mouse homolog of ey in mice can produce functional ectopic compound fly eyes in Drosophila. This suggests that ey is an evolutionarily conserved master gene that begins eye formation. The master role of ey in the development of eyes has led to the question, what are the intermediate genes that ey turns on in the development of the eye?
In this paper, the authors use DNA microarrays to shed some light on the cascade
of proteins that follows expression of ey. To do this, they compare
the arrays of wild-type legs discs to leg discs in which ey is ectopically
expressed during the third larval instar. This is when retinal differentiation
starts, as marked by an indentation of the disc epithelium. This is referred
to as the morphogenetic furrow. The data from this was then compared to the
endogenously expressed genes in eye discs to validate the findings.
This study identified 371 genes that were transcribed in the eye imaginal discs
and ectopically induced by the ey gene. Fifty-five of these genes that
were found in both arrays. When compared to the genes that were expressed in
the eye discs, only some coincided. Some of these were analyzed using in-situ
hybridization. Through these experiments, the authors were able to identify
new genes in the eye development cascade and also show that formerly identified
genes may have additional functions in this cascade.
Materials and Methods:
This section of the paper describes where the Drosophila stocks, DNA microarrays, and targets came from, and how the data was analyzed. The wild-type Drosophila stock came from the iso4BS strain. The experimental stock of flies came from the cross of dppblink-GAL4, recombined with UAS-GAL4, and UAS-ey strains. In this stock, the expression of the ey gene is enhanced in the leg imaginal discs.
The two microarrays used were roDROMEGa and DrosGenome1. These arrays were tested using targets made from the RNA from leg imaginal discs, leg imaginal disks in which an eye field was induced, and eye imaginal disks.
The data analysis is also described here. The average difference(AD) value was used to compare transcript abundance, as a measure of gene expression. A relative scale was set, where genes with AD>/=100 were considered to be expressed, and the AD=20 was the minimal value set to calculate induction folds of all probe sets with AD</=20.
The methods of this experiment are thoroughly and clearly explained. It is clear that the authors thought out the process before hand with controls and checks to ensure the validity of the tests. The fact that they used two separate microarrays for cross-reference lends strength to the findings that are presented later. They also state that they used the same amounts of RNA to create the targets, so the measurements of relative expression should be accurate.
Results and Discussion:
The activity of each gene was measure independently under all three conditions of the study (iso4BS eye discs, iso4BS leg discs, and [dppblink-GAL4, UAS-GAL4/UAS-ey] leg discs). A comparison was done between the genes expressed endogenously in eye discs and in the ey induced expression as a control to distinguish between eye-specific and nonspecific gene expression.
In the section entitled “Discrepancies Between the Two Microarrays,” the authors outline the results and how these can be interpreted. The fact that they used two different microarrays, with different probes, led to unique gene cascades. 371 total genes were identified between the two arrays as expressed in the eye discs and induced ectopically by ey. Of these, 55 of them overlapped (Figure 1A). Of these 55 genes, only 40% of them were also found to be endogenously expressed in the eye discs. Table 1 identifies the 55 genes, highlighting the ones that correspond to ones detected in eye disc libraries by serial analysis of gene expression (SAGE). Table 1 also gives the fold induction values for all the genes in both arrays and scales them by color.
Then in Table 2, the expression of various genes are compared using their AD values. This table shows the discrepancies is expression between the three different testing conditions. These discrepancies are outlined specifically in the text and attests to several contradicting results in gene expression. The fact that some genes are expressed in one microarray and not the other is confusing.
The authors follow up the tables with the in-situ hybridization of some of the genes that were inconsistently expressed in the microarrays, as well as some genes that were not previously associated with eye formation in order to validate some of the new findings. These hybridizations show that some of the genes not expressed in DrosGenome1 are in fact expressed in the eye discs. These genes include Sur-8, sprint, and SP1173. However, the in-situ hybridization does not address cyclin E or sine oculis. ken and barbie is gene that is newly discovered to be associated with eye development through the arrays, and this is confirmed by the in-situ hybridization.
Conclusions:
The conclusion of this paper is that by using microarrays to compare the expression of genes in three different types of imaginal discs, the authors were able to identify some of the genes involved in eye development. This is a conservative conclusion, as it should be. The data that was presented in this paper was spotty and inconsistent in some cases. Many of the tables were listed as published elsewhere, and this made the paper feel unfinished and also made it more difficult to follow.
The one clarifying aspect of the paper was Table 1, which limited itself to the 55 genes found in both arrays. The use of only proteins that showed up in the cross-section of the two arrays seemed logical. However, the next table listed the fold induction values of variously selected genes that did not show up in both arrays. This presentation of the data did not seem to follow the experiment design, but rather seemed like the authors new of some genes so they sought them out to discuss. This arbitrary picking of some genes to do further analysis was confusing.
Then came the in-situ hybridization figures, which legitimized the results of the previous tables. Still, however, only some of the proteins were displayed. There is no mention or attempt at explaining the other contradictory results. The evidence that is presented is compelling, but it would be more so if all the data were shown.
In all, it is understandable why the authors draw such mild conclusions. Some new genes were identified and others were newly associated in the cascade of eye development, which was the goal from the beginning.
Future Work:
The findings of this study lend further support to the role of ey as
an initiator of eye development in Drosophila. However, the fact that
only 40% of the ectopically induced ey genes were also expressed endogenously
means further studies need to be conducted to further elucidate the cascade
events. Also, this study only includes the 55 genes that were found in common
between the two different arrays. The other genes that only showed up in one
array should be more clearly studied to explain how they would not found in
both but do show up in in-situ hybridizations. This study only begins to shed
light on the events that lead to eyes in Drosophila. It is important
that future studies attempt to solidify these preliminary findings.
The fact that other papers have shown that mice and squid homologs also induce
eye formation in the Drosophila, it would be interesting to study whether
these homologs induce the same or similar cascade of events in forming an eye.
For questions or comments, email Heather Maloney