In this study, transgenic fly strains carrying the otd coding sequence or the human Otx2 coding sequence under the control of the heat-inducible Hsp70 promoter were used [24]. Stage 10-17 embryos were given a 25-minute heat pulse in order to overexpress the otd or Otx2 genes and allowed to recover for 25 minutes (see Materials and methods). Ubiquitous overexpression of otd and Otx2 was verified by whole-mount in situ hybridization with otd- or Otx2-specific antisense RNA probes. These experiments demonstrated that RNA was strongly overexpressed 50 minutes after the onset of heat shock in these strains (data not shown). Wild-type control flies were subjected to the identical heat-shock conditions.

Following ubiquitous overexpression of otd or Otx2, transcript profiles were analyzed using a genome-wide high-density oligonucleotide array and compared to the transcript profiles of heat-shocked wild-type control embryos. The transcripts represented on the oligonucleotide array correspond to probe sets that are complementary to approximately 13,400 annotated Drosophila genes according to Release 1.0 of the Drosophila genome [34]. For each experimental condition, several replicates were carried out (see Materials and methods). The degree of reproducibility within individual replicates is shown in scatter plots for four experimental conditions in Figure 1. A complete description of the microarray content as well as all primary data obtained in each individual microarray experiment are available as Additional data files.

An overview of the total number of transcripts that were differentially regulated following otd or Otx2 overexpression is given in Table 1. Two levels of significance for the experimental data are considered in this overview. At a significance level of p ≤ 0.001, a total of 287 genes were found to be differentially regulated following otd overexpression, as compared to heat-shocked wild-type control embryos. This corresponds to 2.1% of the genes represented on the array. At a significance level of p ≤ 0.01, a total of 762 genes were found to be differentially regulated following otd overexpression, as compared to heat-shocked wild-type control embryos. This corresponds to 5.7% of the genes represented on the array. In both cases, approximately a quarter of the differentially regulated transcripts corresponded to known genes, and the rest corresponded to genes that are currently characterized only by sequence information and predicted function (CG-transcripts as described by Celera Genomics [34]).

Overexpression of the human Otx2 gene in Drosophila embryos resulted in a larger number of differentially expressed transcripts than did overexpression of the Drosophila otd gene. At a significance level of p ≤ 0.001, a total of 682 genes were found to be differentially expressed following Otx2 overexpression, as compared to heat-shocked wild-type control embryos. This corresponds to 5.1% of the genes represented on the array. At a significance level of p ≤ 0.01, 1,395 genes were found to be differentially expressed following Otx2 overexpression as compared to heat-shocked wild-type control embryos. This corresponds to 10.4% of the genes represented on the array. Again, in both cases, approximately a quarter of the differentially regulated transcripts corresponded to known genes, and the rest were CG-transcripts.

A subset of the transcripts found to be differentially regulated following otd overexpression were also differentially regulated following Otx2 overexpression. Among the transcripts that were differentially expressed at the significance level of p ≤ 0.001, 93 transcripts were found to be differentially regulated following overexpression of either gene. This implies that 32% of the otd-regulated transcripts were also regulated by Otx2. Among the transcripts that were differentially expressed at the significance level of p ≤ 0.01, 351 transcripts were found to be differentially regulated following overexpression of either gene. This implies that 46% of the otd-regulated transcripts were also regulated by Otx2. In the following, only genes that were differentially expressed at the significance level of p ≤ 0.001 are considered further. We propose that these genes are potential direct or indirect downstream targets for the homeodomain transcription factors otd and Otx2.

When ubiquitously expressed in the embryo, both otd and Otx2 caused a significant transcriptional response of genes encoding a wide variety of functionally different gene products. A detailed classification of the otd- and Otx2-regulated transcripts into different functional classes was carried out according to Gene Ontology (GO) and is presented in Table 2. (In the GO classification scheme, a given gene can be grouped into more than one functional class [35].) The otd- and Otx2-regulated transcripts fall into 92 GO classes, but only about half of these classes are characterized by more than one regulated transcript.

In terms of known function, the two classes with the highest absolute and relative numbers of regulated transcripts were 'enzymes' and 'transcription factors'; this was the case for both otd-regulated and Otx2-regulated transcripts. Other functional classes with high numbers of differentially regulated genes were 'signal transduction', 'DNA binding', 'transporter', 'protein kinase', 'motor', 'ligand binding or carrier', and 'endopeptidase'; again this was the case for both otd- and Otx2-regulated transcripts. Indeed, in most cases in which a functional class was characterized by both otd- and Otx2-regulated transcripts, the relative number (n/M; see Table 2) of otd-regulated transcripts was similar to that of Otx2-regulated transcripts. For example, 2.79% of the otd-regulated transcripts versus 2.20% of the Otx2-regulated transcripts were classified under 'cell adhesion', and 3.48% of the otd-regulated transcripts versus 3.67% of the Otx2-regulated transcripts were classified under 'signal transduction'. Approximately half of both the otd-regulated and the Otx2-regulated transcripts belong to the class 'function unknown'.

Figure 2 shows the otd-regulated transcripts that correspond to known Drosophila transcripts and presents a quantitative representation of the change in expression levels for these transcripts. For clarity, these transcripts are only grouped into mother classes and not into the detailed GO classes. Most of the 63 known transcripts that were differentially expressed following otd overexpression showed increased expression levels; less than 20% of these transcripts were downregulated. The gene with the highest increase in expression level (78-fold) was otd itself, in accordance with our experimental overexpression protocol. Increases in expression levels above 10-fold were also observed for forkhead domain 96cb (fd96Cb), which encodes a nucleic-acid-binding protein, for patched (ptc), which encodes a protein involved in signal transduction, for picot, which encodes a transporter, and for cortactin and Regulator of cyclin A1 (Rca1), which encode gene products of currently unknown molecular function. Only two transcripts showed increases in the 5-10-fold range, namely sugar transporter1 (sut1) encoding a protein involved in sugar transportation, and scraps (scra) encoding an actin-binding protein. The majority of the upregulated transcripts had increases in the 2-5-fold range. The transcript with the most marked decrease in expression was eyegone (eyg), encoding a transcription factor known to be involved in eye development.

Figure 3 shows the Otx2-regulated transcripts that correspond to known Drosophila genes and presents a quantitative representation of their expression level changes. Again, these transcripts are grouped into mother classes and not into detailed GO classes. As was the case for otd overexpression, most of the known transcripts that were differentially expressed following Otx2 overexpression showed increased expression levels. For example, in the functional class of 'enzyme', 45 out of 49 transcripts were upregulated. In total, less than 13% of the 184 Otx2-regulated known transcripts were downregulated. Increases in expression levels above 10-fold were observed for 23 genes and for 6 of these genes, retained (retn), SMC2, licorne (lic), Rtc1, Hairless (H) and deadhead (dhd), the increases were greater than 50-fold. Twenty-two transcripts showed increases in the 5-10-fold range, and, similarly to the otd overexpression situation, increases of 2-5-fold dominated in most of the functional classes. The transcript with the most marked decrease in expression was once again eyg.

Ninety-three transcripts were differentially expressed in response to both otd overexpression and Otx2 overexpression. This indicates that approximately one third of the otd-regulated genes in Drosophila also respond to overexpression of the human Otx2 gene homolog. Figure 4 shows the expression levels for these transcripts, which are likely to represent the common downstream target genes for otd and Otx2. Twenty-one of these transcripts correspond to known Drosophila genes and 72 correspond to annotated CG-transcripts. The expression levels of all of the known transcripts were influenced in the same manner by overexpression of otd and Otx2, in that a given downstream target gene was either upregulated in both cases or downregulated in both cases. Moreover, for most of these transcripts the absolute expression levels were similar in response to otd and to Otx2. Two marked exceptions were pimple (pim), which was upregulated 12.4-fold following Otx2 overexpression and 2.1-fold following otd overexpression, and eyg, which was downregulated 77.6-fold following Otx2 overexpression (but see PCR data below) and downregulated 6.8-fold following otd overexpression. Similarly, the expression levels of 68 of the CG transcripts were influenced in the same manner by overexpression of otd and Otx2. Only in the three remaining cases were transcripts upregulated by overexpression of one of the otd/Otx transgenes and downregulated by overexpression of the other. Thus, approximately one third of the candidate otd downstream target genes in Drosophila are controlled in a comparable manner by the human Otx2 gene homolog.

There are a number of interesting genes among these common candidate genes. The four known transcripts in class 'ligand binding or carrier', scra, Kinesin-like protein at 61F (Klp61F), alpha-Spectrin (alpha-spec) and Centrosomal protein 190 kD (Cen190), are all involved in actin or microtubule binding or movement [36,37,38,39]. This finding is intriguing as one of the Otx2 downstream genes identified in the mouse is a tropomyosin gene, which also encodes an actin-binding protein [40]. Among the four known transcripts in the class 'nucleic acid binding' are the genes Minichromosome maintenance 7 (Mcm7) and Suppressor of variegation 205 (Su(var)205) [41,42] which encode chromatin-binding proteins, and the genes eyg and HLH54F, which encode transcription factors [43,44]. The four known transcripts in the functional class 'enzymes' are Lysozyme D (LysD), cdc2, Rpd3, and BcDNA:LD08534 [45,46,47,48]. Although the cdc2 gene product is classified as 'enzyme', it also acts at the G2/M transition of the mitotic cell cycle [47]. Moreover, Rpd3 encodes a histone deacetylase which is involved in chromatin structure [46]. In the class 'transporter' the n-synaptobrevin (n-syb) gene, encoding a SNAP receptor, is involved in synaptic-vesicle docking and fusion and is expressed in the embryonic CNS [49]. In the class 'signal transducer', the gene EG:30B8.6 encodes a putative GABA-B receptor [50]. Finally, the gene Segregation distorter (Sd), classified as 'enzyme regulator', encodes a Ran GTPase activator [51]. Among the transcripts of known genes are several genes whose precise functional role is not well defined at the molecular level. These are the Bx34 and MRG15 genes [52,53] which encode components of the nucleus and the gluon, Bub3 and pim genes which are all involved in mitosis. gluon encodes a putative component of the condensin complex, and gluon mutants show peripheral nervous system defects during embryogenesis [54]. The gene product of Bub3 is localized to the kinetochore and may function in the mitotic checkpoint [55]. pim is expressed in the embryonic CNS and encodes a protein implicated in mitotic sister-chromatid separation [56].

To confirm the differences in gene expression levels after heat-shock induced overexpression of otd and human Otx2 as compared to heat-shocked wild-type embryos, quantitative reverse transcription polymerase chain reaction (RT-PCR) was carried out on selected candidate target genes. Changes in expression levels were determined for eight genes that were differentially regulated by otd or human Otx2, namely scra, LysD, glu, Rpd3, pim, n-syb, eyg and otd. The genes wunen (wun) and Scc1, whose expression levels remained unchanged in response to otd or Otx2 overexpression, were used as controls. As indicated in Table 3, these experiments showed that the changes in relative expression level, as measured by RT-PCR, are generally consistent with the data obtained with the oligonucleotide arrays. An exception is the data on the response of the eyg gene to Otx2 overexpression; RT-PCR data indicate a weak downregulation (-1.62) whereas oligonucleotide array data indicate a strong downregulation (-77.6).

Cross-phylum gene replacement experiments have shown that the fly otd gene and the homologous human Otx genes are functionally equivalent in vivo, in that overexpression of either gene in Drosophila otd null mutants can lead to the restoration of defects in cephalic and brain development [23,24,25,26]. We have used a combination of transgenic overexpression genetics and functional genomics to gain insight into the equivalence of otd and Otx gene expression in Drosophila at a comprehensive, genome-wide level. Using inducible overexpression and quantitative transcript imaging through oligonucleotide arrays representing the total number of 13,400 currently annotated Drosophila genes, we have identified hundreds of candidate downstream genes for both the fly otd gene and the human Otx2 gene. A comparison of these candidate downstream genes reveals that both otd and Otx genes appear to control an overlapping set of genes; we refer to these genes as common downstream genes. The number of identified common downstream genes for otd and Otx2 depends on the statistical level of significance used to determine whether a given gene showed differential expression in response to transgene overexpression. If the analysis is restricted to highly significant (p ≤ 0.001) datasets, we find 93 common downstream genes, equivalent to 32% of the candidate otd downstream genes or approximately 1% of transcripts in the annotated fly genome. If, in contrast, the analysis is based on significant (p ≤ 0.01) datasets, we find 351 common downstream genes, equivalent to 46% of the candidate otd downstream genes or approximately 3% of transcripts in the annotated fly genome. In either case, a substantial, but far from complete, set of the otd regulated genes are common downstream targets of both fly and human transgenes.

It is interesting that, at the genome-wide transcript level, the Otx2 gene does not appear to be able to replace otd action in full; over half of the transcripts that are influenced by otd overexpression are not influenced by Otx2 overexpression. Given the pronounced differences in amino acid sequence between the OTD and OTX2 proteins, this may not be altogether surprising. The OTD and OTX2 proteins consist of 548 and 289 amino acids, respectively. Shared homology between them is restricted to the homeodomain and to a short domain immediately upstream of the homeodomain as well as a tripeptide at the amino terminus [25]. Moreover, as Otx genes cannot completely replace the otd gene in cross-phylum rescue experiments in vivo, a complete correspondence of otd downstream genes and common otd/Otx downstream genes might