Analysis of the eye developmental pathway in Drosophila using DNA microarrays
Lidia Michaut, Susanne Flister, Martin Neeb, Kevin P. White, Ulrich Certa, and Walter J. Gehring
Journal Article Review
Summary of Purpose and Process
This paper concerns the genetic cascade controlling eye morphogenesis by studying ectopic eye in the developmental stage in Drosophila when cells start differentiating into photoreceptors. By using full genome microarrays, wild-type gene expression was compared to the ectopic expression of the eyeless gene, in leg discs. The eyeless gene encodes one of two Drosophila Pax-6 genes that encodes transcription factors that are considered capable of activating the cascade that causes eye formation. The high degree of conservation of Pax-6 is illustrated by its ability to encode cascade starting transcription factors for both the vertebrate camera-type eye and the insect compound eye.
The third larval stage in Drosophila is important in this investigation because this is where retinal differentiation starts to occur. The imaginal disc epithelium indents creating a morphogenetic furrow, so termed because the cells within this furrow begin to differentiate after arresting in G1 phase, while the undifferentiated cells anterior to the furrow divide asynchronously. The furrow cells either, become grouped into preclusters of photoreceptors or undergo a second mitotic wave before becoming differentiated photoreceptors: pigment and cone cells. The two Drosophila Pax-6 genes are expressed in the eye precursors but can lead to ectopic eye formation on adult appendages when expressed in other imaginal discs. (paper) (picture)
One of the goals was to identify genes that were up-regulated when an eye morphongentic field was induced. Wild-type leg discs were compared to leg discs where ey (eyeless) is ectopically expressed by using DNA microarrays. Genes from the ectopically expressed eyes were then compared to genes in endogenously expressed eye discs. These researchers identified 371 such genes, some of which had not previously been associated with eye formation.
The microarry comparison used two different sets of oligonucleotide probe sequences to arrive at results. These two probe sequence sets came from independently designed Drosophila full-genome high-density oligonucleotide arrays: roDROMEGa and DrosGenome1. Both arrays represented some genes by more than one probe set to include different splice variants. Thus, some genes are represented more than once on a given array. Conversely, some genes were represented on only one of the two arrays. Drosophila strain iso4BS was used as wild-type strain. For the ectopic expression of ey, dppblink-GAL4 was recombined with UAS-GAL4 (upstream activating sequence-GAL4) and crossed to UAS-ey. cRNA from imaginal disks was fragmented and hybridized to the arrays. These researchers found the average distance (AD), values between expression signals, were coupled in 3-5 biological replicates to find the P-value of an unpaired t-test. P represented the probability at which the null hypothesis was rejected. The null hypothesis was that no difference in the expression of a given gene exists between experimental samples.
Summary of Figures
Figure 1A is an illustration of the results from the microarrays. Of the 371 genes induced by ey and expressed in the eye disks, 228 were detected using the roDROMEGa microassay and 198 were detected using the DrosGenome. 55 of genes in roDROMEGa were the same as those in DrosGenome. This is the figure that establishes that there are 371 unique ey-induced genes because the genomic DNA was probed with oligonucleotides that complimentary base paired Drosophila genome downstream of ey.
Figure 1B classifies 254 of the ey-induced genes, for which molecular function is known. Over 40 are enzymes and the next largest category is transcription factors. ey is known to encode Pax-6 transcription factors. This horizontally aligned histogram suggests that much of the area downstream from the ey gene encodes for transcription factors also.
Table 1 lists the 55 genes, induced by ey and expresed in the eye discs during ectopic eye development, that were detected by both the arrays. This is a tabultion of serial analysis of gene expression and highlights, in blue, the genes that had similar tags detected in eye disc libraries. There are two big columns within each of which there are four smaller columns. Of these smaller columns, the first column is the gene and the second column the molecular function of the protein that the gene encodes for. The last two columns compare the fold induction (IF) of the two assays. There are five genes for which IF values agree exactly. However there is a difference between the IF values for most genes. For CG140595 the IF values differ by 43.1. The main thing to note is that ey has a strong IF as assessed by both assays.
Table 2 compares the wt and ectopically expressed leg discs with the IF of the eye imaginal discs. Figure 2 shows ey-induced expression as it naturally occurs in wt eye discs. It is an in situ hybridiztion probed with RNA that is antisense to specific genes. Ultimately, this figure shows where in the wt eye discs each protein is expressed. Each protein was one that had been anaylyed in a table. The genes that express a stronger signal when probed In Situ: ken, gh11415, CG11849 and CG13651, were found to function directly in eye development, for example, CG11849 is a DNA-binding domain and ken is a transcription factor.
This paper reports and presents some significant data. It employs efficient method of microarrays to further research the influence of the eyeless gene. Figure 1A is sufficient to suggest that there are 55 ey induced genes that warrent further investigation. The functional assessment of genes in Figure 1B would be important if there were more data to support the formulated thought of the authors. Even if the purpose of each ey induced gene correctly corresponds to this histogram, there are 117 genes whose functions are unknown.
The authors acknowledge the discrepencies between the two microarrays and give possible reasons for discrepencies including probe selection parameters used to design the two arrays and noise in the MAS 4.0 alogrithm used to find average differences between expressed signals. However, the authors do not rework experimental designs before progressing. Thus the tabulated data is largely open to interpretation due to the discrepencies between the two microarrays.
Figure 2 is a clear comparison of where different genes occur in endogenous, ey-induced, wt, eye discs but it is unclear why these thirteen genes were picked.
Finally, this paper is well researched in the sense that it sites a lot of other literature and follows a natural progression of previous projects, however, it does not generate much new data itself.
First, it is important to reconcile the differences generated from the two microarrays. It might be beneficial to run the same experiment again using the same roDROMEGa and DrosGenome1 microarrays with more of the original probes so as to ascertain whether the system was contaminated in some way. If the outcome was similar to those results seen in figure one, then it might be possible that the RNA is degrading and probes of DNA could be employed.
Along with microarrays, Drosophila are a powerful tool and the best way to determine molecular function of a gene might be to create a construct, in Drosophila, that lacks it. This could hypothetically be done for all 371 genes, but it must be noted that since the formation of the eye is said to be a cascade, some of those 371 genes are most likely dependent on others trascribed downstream of ey.
Eventually, since the Pax-6 genes are evolutionarily conserved, it might be beneficial to investigate the genes of other species that are transcribed in the eye imaginal discs and indueced by ey during ectopic formation.
Michaut, Lydia. et. al. 2003. Analysis of the eye developmental pathway in Drosophila using DNA microarrays. 100 (12): 4024-4029.
Hadler, Georg. et. al. 1995. Induction of Ectopic Eyes by Targeted Expression of the eyeless Gene in Drosophila. <http://bio.davidson.edu/Courses/Molbio/restricted/Eyepaper/eye.html>
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