The homeotic/Hox genes encode a network of evolutionarily conserved homeodomain transcription factors that are involved in the specification of segmental identity along the anterior-posterior body axis of animals as diverse as insects and vertebrates [1,2,3,4,5,6]. In Drosophila, these genes are arranged on the chromosome in two gene clusters known as the Antennapedia and Bithorax complexes. There is a correlation between the relative position of the Hox genes within the cluster and their spatial and temporal expression pattern in the body in that genes located towards the 3' end are expressed more anterior and earlier than genes located towards the 5' end (spatial and temporal colinearity) [7,8,9,10,11].

Given their central role in developmental processes, it has been proposed that the homeoproteins do not act directly to specify morphological differences but rather control a battery of subordinate genes encoding cellular functions directly required in differentiation [12,13]. In search of these subordinate genes, various strategies such as enhancer trapping, immunoprecipitation of chromatin fragments, subtractive hybridization, selection for binding sites in yeast, and heat-shock-induced overexpression have been used [9,14,15,16,17,18,19,20,21].Only a small number of target genes of homeoproteins have been identified to date, however; most of these encode either transcription factors or cell-signaling molecules [9]. In contrast to these results, recent studies suggest that homeoproteins may bind at significant levels to the majority of genes in the Drosophila embryo and regulate a large number of downstream genes [22,23].

Here we focus on the homeotic gene labial (lab) in the Drosophila embryo. It is the most proximal gene within the Drosophila Antennapedia complex; it encodes an Antennapedia-like Q50 homeodomain transcription factor and is one of the most anteriorly expressed homeotic genes along the anterior-posterior body axis [24,25,26,27]. Genetic studies have demonstrated that lab is required for proper head formation [28] and for the specification of cellular identity in the midgut [29] as well as in the embryonic brain [30]. The lab gene and its vertebrate Hox1 orthologs are among the best-characterized examples of evolutionary conservation of structure, expression and function of Hox genes in animal development [31,32,33,34,35].

To address the question of which and how many downstream genes are under control of lab, we used a combination of in vivo overexpression techniques and quantitative transcript imaging with oligonucleotide arrays. By using transgenic flies carrying the lab gene under the control of a heat-inducible promoter, we ubiquitously overexpressed lab following heat-shock treatment in Drosophila embryos. We then used high-density oligonucleotide arrays representing 1,513 identified Drosophila genes for large-scale detection and quantification of induced gene expression [36,37,38,39]. We find significant changes in gene expression for 96 identified genes following lab overexpression. Quantitative reverse-transcriptase PCR on a selection of these genes verified the differential expression levels in response to heat-shock-induced overexpression of lab. Our findings identify a number of novel candidate downstream genes for lab and thus show that oligonucleotide arrays are powerful tools for analyzing, at a genome-wide level, the number, identity and quantitative expression level of genes in the Drosophila embryo.

In this study, transgenic fly strains carrying the lab coding sequence under control of the heat-inducible Hsp70 promotor were used [40]. Stage 10-17 embryos were given a 25 minute heat pulse to overexpress lab, and allowed to recover for 25 minutes (see Materials and methods for heat-shock protocol). Ubiquitous overexpression of lab was verified by whole mount in situ hybridization with a lab-specific antisense RNA probe. Ubiquitous overexpression of Labial protein (Lab) was verified by immunocytochemistry with an anti-Lab antibody. These experiments demonstrated that both lab RNA and Lab protein were strongly overexpressed 50 minutes after the onset of heat shock in these strains (Figure 1). Wild-type control flies were subjected to an identical heat-shock regime.

Following ubiquitous overexpression of lab, transcript profiles were analyzed using a high-density oligonucleotide array and compared to the transcript profiles of heat-shocked wild-type control embryos. For each of the two experimental conditions ('hs-wt'and 'hs-lab'), four replicate experiments were performed and the data set was analyzed with an unpaired t-test (see [39] and Materials and methods). The genes represented on the oligonucleotide array correspond to probe sets that are complementary to 1,513 identified and sequenced Drosophila genes. Most of these genes can be grouped into 14 functional categories according to the nature of the encoded protein [39].

At a significance level of p ≤ 0.01, a total of 96 genes were found to be differentially regulated following lab overexpression compared with heat-shocked wild-type control embryos. This corresponds to 6.3% of the genes represented on the array. At a significance level of p ≤ 0.05, 205 genes were found to be differentially regulated following lab overexpression compared with heat-shocked wild-type control embryos (data not shown). This corresponds to 13.5% of the genes represented on the array. The relative distribution of lab-regulated genes in particular functional classes, as well as the percentage of genes regulated within a given functional class, were comparable between the p ≤ 0.01 group and the p ≤ 0.05 group. Only genes that were differentially expressed at a significance level of p ≤ 0.01 are considered further. We propose these genes to be potential direct or indirect downstream targets for the homeodomain transcription factor Labial.

When ubiquitously expressed in the embryo, lab caused a significant transcriptional response among a wide variety of genes belonging to all functional classes represented on the array (Table 1). The functional class with the highest absolute number of differentially regulated genes was 'transcriptional regulation' (n = 20). Other functional classes with high numbers of differentially regulated genes were 'metabolism' (n = 13), 'proteolytic systems/apoptosis' (n = 12), 'cell-surface receptors/cell adhesion molecules (CAMs)/ion channels' (n = 12), and 'RNA binding' (n = 7). Relative to the number of genes represented on the array within a given functional class, the highest relative percentage of differentially regulated genes was found in the functional classes 'proteolytic systems/apoptosis' (19.4%), 'cell cycle' (13.5%), 'transposable elements' (11.4%), 'chromatin structure' (11.1%), 'RNA binding' (11.9%), and 'transcriptional regulation' (7.6%).

Figure 2 shows the lab-regulated genes and presents a quantitative representation of the change in expression levels for these genes. Of the 96 genes that were differentially regulated, 48 showed increased expression levels and 48 showed decreased expression levels. The gene with the highest increase in expression level (26-fold) was lab itself, in accordance with our experimental procedure. Increases in expression levels above 10-fold were also observed for Bicaudal C (BicC), swallow (swa) and oskar (osk), all encoding proteins involved in RNA binding, as well as for the wings apart-like (wapl) gene belonging to the functional class 'chromatin structure'. The increased expression levels in BicC, swa, and osk are surprising, as all these genes are known to function as maternal control genes during early embryogenesis [41,42]. As lab activity is normally only observed from gastrulation onwards [26], this suggests that high levels of widespread ectopic lab expression are able to activate genes which under wild-type conditions show spatio-temporal expression domains that do not overlap with that of lab.

Increases in the 5-10-fold range were seen for six genes. One encodes the enzyme ubiquitin carboxy-terminal hydrolase, whose mammalian homolog has also been found to be differentially upregulated by ectopic overexpression of the lab ortholog Hoxa1 [43]. Increased expression levels in the 1.5-5-fold range were prominent in several functional classes. For example, in the functional class 'proteolytic systems/apoptosis', 12 of 13 differentially regulated genes were upregulated and most of these showed increased expression levels ranging between 1.5 and 5. Strikingly, in the functional classes 'cell cycle' and 'transcription/replication/repair' all the differentially regulated genes were upregulated. Thus, differentially expressed genes such as twine (twe), Cyclin B (CycB) and Cyclin D (CycD), belonging to the functional category 'cell cycle', were all upregulated following lab overexpression. It is notable in this respect that recent experiments carried out on mammalian cell lines showed that ectopic overexpression of the lab ortholog Hoxa1 also causes differential upregulation of cell-cycle regulatory proteins [43].

Decreases in expression levels in the 10-fold and above range were not observed, and decreases in the 5-10-fold range were only seen for the transposable R2 rDNA element gene. Decreased expression levels in the 1.5-5-fold range were, however, prominent in the functional class 'transcriptional regulation' and in the functional class 'cell-surface receptors/CAMs/ion channels'. Thus, almost three-quarters of the differentially regulated genes encoding transcription factors showed significant decreases in expression levels following lab overexpression. For example, the genes prospero (pros), Distal-less (Dll), tailup/islet (tup), mirror (mirr), huckebein (hkb) and abrupt (ab) were all downregulated. Interestingly, it has been shown that Distal-less is a direct target of homeotic gene control [9], and recent genetic studies demonstrated that tailup/islet expression in the lab-specific territory of the embryonic Drosophila brain is dependent on lab gene action [30]. As with the functional class 'transcriptional regulation', 10 out of 12 genes representing the functional category 'cell-surface receptors/CAMs/ion channels' were downregulated, including the genes derailed (drl), frizzled 2 (fz2), Neurotactin (Nrt), Neurexin (Nrx), rhomboid (rho) and 18 wheeler (18w). As is the case for tailup/islet, Neurotactin expression in the lab-specific territory of the embryonic Drosophila brain is dependent on lab gene action [30]. The 18w locus has been identified as a binding site of the homeotic protein Ubx in polytene chromosomes [18].

To verify the differences in gene expression level after heat-shock-induced overexpression of lab as compared to heat-shocked wild-type embryos, quantitative RT-PCR was performed on selected candidate target genes. Changes in expression levels were determined for eight genes that were differentially regulated following lab overexpression, namely lab, swa, Ubiquitin conjugating enzyme 4 (UbcD4), twe, cycB, Ubiquitin carboxy-terminal hydrolase (Uch), scratch (scrt) and phosphoenolpyruvate carboxykinase (Pepck). The gene squid (sqd), whose expression level remained unchanged under both experimental conditions, served as a control. As indicated in Table 2, these experiments showed that the changes in relative expression level, as measured by RT-PCR, are consistent with the data obtained with the oligonucleotide arrays.

We have used a novel combination of manipulative genetics and functional genomics to gain further insight into homeotic gene action in Drosophila from a genomic perspective. Using inducible overexpression and quantitative transcript imaging through oligonucleotide arrays, we have identified 96 genes (only 6.3% of the 1,513 identified genes represented on the oligonucleotide array) whose expression levels change significantly following lab overexpression.

These findings suggest that Lab regulates a limited and distinct set of candidate downstream genes. This appears to contrast with previous reports indicating that in late embryogenesis the majority of Drosophila genes are under control of homeoproteins [23,44]. It should be stressed, however, that a number of features of our functional genomic analysis prevent a direct comparison with these reports, which are based on DNA-binding studies. First, although our analysis can quantify gene expression accurately and simultaneously for many identified genes, the temporal and spatial resolution of our analysis is low. This is because our experimental design averages gene expression throughout the embryo and during several embryonic stages. In consequence, our analysis may fail to detect genes that are only expressed in a small subset of cells or during a very restricted time period in embryogenesis. Second, our overexpression protocol makes it difficult to control the level of Lab protein as well as the temporal dynamics and stability of this protein. As different levels of a given homeoprotein can have different functional consequences in terms of developmental specificity [29,45], the high level of Lab protein may bias the set of candidate downstream target genes identified. Third, in our studies lab overexpression is not accompanied by concomitant overexpression of cofactors, which are thought to act together with homeotic proteins to determine their in vivo target specificity [34,46]. It should be noted that the gene mirror, which has been proposed to be an additional cofactor for homeoprotein specificity [47], was detected as downregulated following lab overexpression.

Although the question of the total number of target genes that are regulated by homeoproteins in vivo must await further analysis, our genomic perspective of lab gene targets does reveal several specific features of homeoprotein action. First, our results demonstrate that the homeodomain transcription factor Lab acts on numerous candidate target genes that also encode transcription factors. The category 'transcriptional regulation' comprises one of the largest sets of differentially regulated genes following lab overexpression. This is consistent with the idea that homeobox genes establish developmental patterns by acting through a cascade of transcription factors which regulate the expression of their own subset of downstream genes [1,2,9,15]. Second, our data indicate that upregulation of gene expression is prominent in several functional classes. Thus, virtually all of the lab-regulated genes in the functional classes 'cell cycle', 'transcription/replication/repair', and 'proteolytic systems/apoptosis' show increased expression. Third, our results show that lab overexpression causes not only widespread activation but also widespread repression of gene expression. Thus, of the 96 genes that are potential targets of lab, half are downregulated by overexpression of this homeobox gene. This widespread repression is especially pronounced in the functional classes of 'transcriptional regulation' and 'cell-surface receptors/CAMs/ion channels'. For example, following lab overexpression, over 80% of the differentially regulated genes encoding cell-surface receptors/CAMs/ion channels showed decreased expression.

Taken together, our results identify a large number of novel candidate downstream genes of the homeodomain transcription factor Lab. To our knowledge, most of these 96 identified and sequenced genes have not been previously shown to be lab targets. At present, we do not know which genes are direct targets (regulated directly by Lab protein binding to DNA regulatory sequences) or indirect targets of lab gene action. Furthermore, our results demonstrate that oligonucleotide arrays are useful tools for analyzing, at a genome-wide level, the number, identity and quantitative expression levels of candidate downstream genes differentially regulated in vivo by developmental control genes. This confirms the general utility of microarrays for studying diverse molecular and cellular processes in Drosophila [48,49,50]. Considering the evolutionary conservation of gene structure, expression and function [1,35], we propose that these results obtained in Drosophila will also be valid for lab orthologs in other animals, including vertebrates. It will now be important to determine which of the detected candidate downstream genes in Drosophila are direct targets and how they exert the developmental genetic programs imposed by lab gene action.