Here, genes that underlie developmental changes that occur during metamorphosis were identified by assaying gene expression in male and female wild type animals and animals that lack germline tissues, using a two-color, glass-slide microarray approach (see Methods; [9
]). The wild type animals are the Canton S (CS) strain and the animals that lack germline tissues are the progeny of female flies homozygous for the maternal-effect, recessive mutation tudor
), hereafter referred to as tud
]. Experimental samples were compared to a common reference sample consisting of RNA derived from male and female CS pupae collected from all stages of metamorphosis; this approach facilitated comparisons across all the experiments (see Table for experimental design). Gene expression was assayed at five time points in animals collected every 24 hours, ranging from 0 hours after puparium formation (APF; 0 hour APF is the white pre-pupal stage) to 96 hour APF (pharate adults).
Microarray experimental design for time course and sex hierarchy experiments.
Additionally, somatic, sex differences in transcript abundance for genes regulated downstream of dsx
(Figure ) were determined at a mid-pupal stage (48 hour APF). Microarray comparisons using RNA from the following genotypes were performed: wild type males and females from two different strains (CS and Berlin), male and female tud
progeny, wild type females and tra
pseudomales, and wild type females and dsxD
pseudomales are chromosomally XX animals that produce DSXM
, the male-specific isoform of DSX, and as a result look phenotypically similar to wild type males [11
]. The analyses of two distinct mutant genotypes that produce DSXM
in a chromosomally XX background facilitated the identification of genes that are sex-differentially expressed downstream of DSX, as opposed to differences in sex-chromosome content, and together with the analyses of two different wild type strains, reduced the identification of genes for which differential expression is due to differences in strain, or genes acting only upstream of dsx
in the sex hierarchy. Additionally, gene expression was compared between intersexual male and female flies that do not produce DSX (dsx
]) and wild type males and females, respectively, to examine the modes of dsx
-regulated gene expression (see Table ).
Time course experiment: gene expression during metamorphosis
The expression data from both wild type and tud
progeny males and females was first analyzed to identify genes expressed in somatic and germline tissues during metamorphosis. Here, 8,482 and 9,725 genes of the 13,820 genes examined had expression data in the tud
and wild type experiments, respectively, demonstrating that ~70% (9,725 of 13,820 genes in wild type flies) of the predicted Drosophila genes are expressed during metamorphosis (see Methods for details). This also suggests that approximately 1,200 additional genes are expressed during metamorphosis due to the presence of the germline in wild type males and females. Our previous study examining gene expression in wild type flies found a larger percentage of genes (~94% of genes represented on arrays) expressed during metamorphosis (3,784/4,028 genes; [7
]). Our previous study employed a cDNA microarray platform representing about one-third of the genes in Drosophila
. As such, it was biased for genes with high expression levels, which might account for the differences in the two studies [7
Somatic sex-differential gene expression during metamorphosis
To identify genes whose transcript abundances differ between the two sexes in somatic tissues through metamorphosis, the tud
progeny gene expression data was analyzed using F-statistics, conducted using LIMMA contrasts with sex and time as independent factors (see Methods and Additional file 1
, for details). For the F-test analyses, lists of P
values were converted to q
values, an estimate of the false discovery rate [13
]. Two-hundred-fifty-eight genes were identified with significant somatic, sex-differential expression (q
< 0.15 for sex or sex-time interaction term; see Methods for details; Additional files 2
). Similar numbers of genes were identified with male- and female-biased expression (124 and 134, respectively). Overall, the percentage of genes with somatic, sex-differential expression during pupal stages (1.9%) is similar to the number of genes displaying somatic, sex-differential expression at adult stages (1.7% of genes [68/4028] in [7
], 2.5% genes [301/11893] in [14
], and 1.4% of genes [167/11893] in [15
]). In contrast, thousands of genes show sex-differences in transcript levels in the male and female germline tissues, at both pupal and adult stages (see germline section below; [7
For the 258 genes with somatic, sex-biased expression, moderated-t
-tests were performed [16
], comparing gene expression in tud
progeny males and females to determine at which stage the gene displays somatic, sex-differential expression (Table ). The five time points (0, 24, 48, 72 and 96 hour APF) do not have large differences in the numbers of genes with somatic, sex-biased transcript levels (q
< 0.15; 75, 181, 79, 131, and 152 genes, respectively; Figure ), with the data from the 24 hour time point containing the largest number of genes.
Sex differentially expressed genes in somatic tissues during metamorphosis.
Figure 2 The number of genes with somatic sex-differential transcript levels differs across metamorphosis. The abscissa indicates the five time points during metamorphosis examined (0, 24, 48, 72, and 96 hour APF). The ordinate indicates the number of somatic, (more ...)
At the statistical threshold used for the t
< 0.15), 242 of the 258 genes identified by F-tests showed significant, somatic, sex differential-expression at a minimum of one time point. Close to half of these genes (119 genes) displayed somatic, sex-differential expression at only one or two time points, suggesting that they are likely to mediate discrete, sex-specific, developmental events. RNA on the X 1
) is male-biased and one of only thirteen genes that were sex-differentially expressed at all five time points examined. roX1
and a gene RNA on the X 2
) produce non-coding RNAs that are components of the dosage compensation macromolecular structure [17
was either expressed exclusively in males or four times higher in males than females at each of the five time points examined. Dosage compensation is the process in which genes on the single X chromosome in males undergo increased transcription, which results in roughly equal amounts of mRNA product produced by the two X chromosomes in females [reviewed in [18
]]. Of the additional 12 genes (three and nine with male- and female-biased expression, respectively) with sex-differential expression at all five time points examined (Microsomal glutathione S-transferase-like
, Glutathione Synthetase
, Larval serum protein 1 alpha
, Ilp6, CG1702
, Succinate dehydrogenase B, CG7430
, and cabut
), eight are located on the X chromosome. Sex-lethal
), the gene at the top of the sex-determination hierarchy (see Figure , [reviewed in [3
]]), displays female-biased expression at four of the five time points examined. Interestingly, CG3056
, a gene with male-biased expression at four of the five time points, is a paralog of Sxl
]; the product of this gene may underlie additional sex-differential splicing that regulates sex-specific development.
The differences in transcript abundances observed at each stage are due to biological differences and not poor quality data, as the microarray data showed high correlation among experimental replicates and similar numbers of genes had expression data at each time point in both sexes (see Additional file 4
for all microarray correlations and number of genes expressed in each experiment).
Time course experiment: cluster analyses
One of the primary goals of this study was to identify genes that direct the patterning and morphogenesis of sexually dimorphic, somatic tissues. Hierarchical clustering, an algorithm that groups genes based on the similarity of their expression profiles [20
], was performed using the five time points of tud
progeny expression data to identify genes with similar expression profiles in somatic tissues. Because the numbers of expected clusters were unknown, other clustering methods, including K-means clustering and self-organizing maps [reviewed in [21
]], were not employed. Thirty-eight clusters, each with a greater than 0.80 average Pearson's correlation in gene expression profiles and containing 15 genes or more, were identified and further analyzed (Figure and Additional file 5
). Combined, these clusters contained 4410 genes, including 82 genes with alternative transcripts expressed in two clusters and four genes with alternative transcripts expressed in three clusters. There is a high degree of separation among the clusters, with an average correlation of 0.026 between all of the clusters, demonstrating that the expression profiles for genes within a cluster are not similar to expression profiles for genes in other clusters and thus each should be considered separately in this study (see Methods).
Figure 3 Clusters of genes with similar expression profiles in somatic tissues during metamorphosis. Clusters were generated using gene expression data from male and female tud progeny at five time points during metamorphosis, indicated at top of clustergram. (more ...)
An analysis of the expression data across the five time points for the genes in each of the 38 clusters identified 51 peaks and 49 troughs of gene expression within the clusters (Figure ; Methods). Based on a re-sampling analysis, each of these 51 peaks and 49 troughs was significantly different than the average expression at all time points in the cluster from which they were identified (P
< 0.01; see Methods). When wild type expression data was incorporated into the cluster analyses, gene expression profiles appear similar in wild type and tud
progeny experiments, based on visual inspection (Additional file 6
); only genes with expression in tud
progeny were included in the cluster analyses. Furthermore, a statistical comparison of the average tud
progeny expression data to the average wild type expression data within each cluster showed that 31 of the 38 clusters had correlations >0.70, demonstrating a high degree of similarity of expression for genes in tud
progeny and wild type animals. The seven clusters with correlations <0.70 between the average tud
progeny and wild type expression data contained only 172 of the 4410 genes present in the 38 clusters. Therefore, for most genes examined, the pattern of gene expression in somatic tissues during metamorphosis does not appear to be largely influenced by the presence of the germline.
To functionally analyze these clusters, the sets of genes from each cluster were examined using the program DAVID, which identifies overrepresented functional groups among the genes in each cluster, as compared to all the genes represented on the array platform (see Methods; Additional file 7
; DAVID is the Database for Annotation, Visualization and Integrated Discovery [22
]). To confirm the DAVID results, an independent tool that searches for enrichment of Gene Ontology terms (FlyMine; [23
]) was used to assess overrepresented functional categories and gave very similar results (see Methods for details).
Gene expression during metamorphosis
At the white pre-pupal stage (wpp; 0 hour APF), the fly is transitioning from a wandering larva into an immobile pre-pupa. The pre-pupal animal initiates the major larval-to-adult transition in several discrete ways: 1) strictly larval tissues are destroyed and replaced by corresponding adult tissues [reviewed in [24
]], 2) imaginal discs and rings begin to give rise to adult structures including eyes, antennae, wings, legs, and genitalia [reviewed in [25
]], 3) histoblast nests proliferate in number and give rise to non-imaginal disc derived adult epidermal structures [26
], and 4) the larval central nervous system is remodeled through the destruction of some larval neurons, proliferation of neuroblasts to generate new neurons, and remodeling of some larval neuronal projections [reviewed in [27
]]. The 24 hour APF time point is between the pre-pupal pulse of ecdysone, which peaks at 12 hour APF and triggers head eversion, and the large pupal pulse of ecdysone that initiates around 24 hour APF [reviewed in [1
]]. By 24 hour APF the majority of larval-specific tissues are degraded and adult development is triggered [reviewed in [28
]]. During the time between the 24 to 48 hour APF stages, the imaginal discs are still undergoing morphogenesis, but are close to their final adult form. The wings, leg muscles, abdominal bristles, abdominal muscles and internal genital ducts are all well formed, while further development of the eyes, legs, wings, thorax, and abdomen is occurring [reviewed in [2
]]. During the later stages of metamorphosis (72 hour APF), many of the tissues and structures developing in the pupae are close to their final adult form [reviewed in [27
]]. By 96 hour APF, the pupa is within a few hours of eclosion, or emergence of the adult fly [reviewed in [30
To understand the transcriptional basis of these complex developmental events, expression data was analyzed in the following ways: first, genes with similar expression patterns in both male and female somatic tissues were identified based on the hierarchical cluster analyses (Figure ). Second, clusters were identified that contained genes that either had a peak or trough of their transcript abundance at each time point (see Methods). At the 0 hour APF stage, Cluster 5 (586 genes) has genes with peak expression and was enriched for genes that encode proteins that function in the proteosome (34 genes; P = 6.8 × 10-31), have cell death activities (35 genes; P = 4.2 × 10-7) or peptidase activities (90 genes; P = 5.4 × 10-18) and thus likely function in the histolysis of larval tissues. Cluster 21 (254 genes) has genes in a trough of expression and is enriched for genes whose products function in development (60 genes; P = 4.2 × 10-6), differentiation (25 genes; P = 4.6 × 10-4), and cell communication (54 genes; P = 9.8 × 10-5), suggesting that a large fraction of genes that function in these patterning and developmental processes are at low transcript levels immediately after pre-pupal formation.
At the 24 hour APF stage, the largest cluster identified, Cluster 13 (1,121 genes), shows peak transcript abundance (Figure ). Cluster 13 is overrepresented with genes encoding products that are annotated as functioning in imaginal disc morphogenesis (73 genes; P = 2.1 × 10-15), neurogenesis (58 genes; P = 3.9 × 10-14), programmed cell death (62 genes; P = 9.9 × 10-11), nervous system development (122 genes; P = 2.4 × 10-18), and transcription (183 genes; P = 2.8 × 10-16), demonstrating that at about 24 hour APF, many genes that drive morphogenesis and patterning have reached a peak in their transcript abundance, marking this period as critical for patterning and morphogenesis.
At the 48 hour APF stage, Cluster 18 and Cluster 21 contain 138 and 254 genes, respectively, and show peak levels only at this stage (Figure ). Cluster 18 is enriched with genes whose products function in cell organization and biogenesis (21 genes; P = 0.016), appendage morphogenesis (5 genes; P = 0.032), and pupal development (8 genes; P = 0.041), suggesting that although the rudimentary adult structures are formed, there are still many structural changes taking place. Consistent with this idea, Cluster 21 is enriched with genes whose protein products function in development (60 genes; P = 4.2 × 10-6), cell communication (54 genes; P = 9.8 × 10-5) and morphogenesis (30 genes; P = 1.1 × 10-4).
At the 72 hour APF stage, genes in Cluster 31 (737 genes, Figure ), which are in a trough at the 0 and 24 hour APF stages, quickly increase in transcript levels to ultimately peak at 72 hour APF. Cluster 31 is overrepresented with genes that encode products that function in the mitochondria (153 genes; P = 1.4 × 10-77).
One large cluster, Cluster 32 (278 genes; Figure ) contains genes that showed a sharp rise in transcript levels at 96 hour APF, but no peaks early in metamorphosis. This cluster is enriched for genes encoding products that function in the response to light stimulus (7 genes; P = 6.6 × 10-4), visual perception (7 genes; P = 0.0050), and rhabdomere function (6 genes; P = 3.0 × 10-5), all of which are critical for proper vision and development of the adult eye. On the other hand, the largest cluster, which is enriched for genes that encode products functioning in developmental processes (all P = 3.9 × 10-14) and peaked in transcript abundance at 24 hour APF, is in a trough of transcript levels at 96 hour APF (Cluster 13, 1,193 genes), consistent with the idea that morphogenesis is largely complete by the pharate adult stage.
Sex-biased gene expression in germline tissues during metamorphosis
Genes with expression during metamorphosis in either male or female germline tissues were identified using two independent F-statistic analyses (q
< 0.15 for each test; see Methods; see Additional file 2
for numbers of genes with significant expression differences). This statistical approach also identifies genes whose expression in somatic tissues is dependent on the presence of male or female germline tissues, respectively; these two classes of genes cannot be distinguished in this study. The gene sets that are expressed within or dependent on the presence of the germline in males and females are referred to as the pupal male- and female-biased germline sets, respectively. Sets of 659 and 342 genes with male- and female-biased expression in the germline, respectively, were identified (Additional files 8
). Both the male- and female-biased pupal germline sets had significant overlap with genes previously identified as highly expressed in adult male and female germline tissues, respectively (P
< 1.1 × 10-4
, hypergeometric test for both sets; [14
]). Five-hundred-forty-three genes identified in the pupal male germline set were on the previous study's array platform [14
]. Of those 543 genes, 392 were highly expressed in the adult male germline. Similarly, 305 genes identified here as being expressed in the pupal female germline were present on the previous study's array platform. Forty-seven of those 305 genes were also highly expressed in the adult female germline.
Gene expression in the male germline has already initiated at the start of metamorphosis, and by 24 hour APF has reached its peak level of gene expression; this high level of expression lasts throughout metamorphosis (Figure ). Sixty-nine of the 659 genes in the pupal male germline set encode products that function in the mitochondria (P
= 5.8 × 10-19
; DAVID analysis), consistent with the essential role for mitochondria in spermatid development and adult function [reviewed in [31
]]. One-hundred-fifty-one genes (of 543 genes, 28%) were identified that are expressed in the male germline during metamorphosis, but were not previously identified as expressed in the adult male-germline (present on platform of previous study, but not significantly differentially expressed [14
]), suggesting that there is pupal-specific, male-germline gene expression that might underlie male germline development.
Figure 4 Expression profiles of genes expressed in the male and female germline during metamorphosis. Expression profiles were generated by averaging the gene expression data of all genes that have high expression in the male or female germline at each time point. (more ...)
Previously, it was observed that most genes expressed in the female germline showed the first post-embryonic peak of transcript abundance during adult stages [7
]. However, because our previous study did not have data from pupal stages examining gene expression in each of the sexes separately, or in male and female tud
progeny, we were unable to definitively identify the genes expressed in the female germline at pupal stages. The data presented here demonstrate that are a substantial number of genes with female-biased germline expression during pupal stages (Figure ). Between the 48 and 72 hour APF stages, the structures derived from the female genital disc establish connections with female gonadal tissues to form the female reproductive system [reviewed in [32
]]. The development of female reproductive structures likely requires gene expression in both somatic and germline tissues. This idea is consistent with the functions of genes with pupal female germline expression, as this set is overrepresented with genes that function in developmental processes (76 genes, P
= 0.0031; DAVID analysis).
Interestingly, the transcript level of genes expressed in the pupal female-germline also peaks in both wild type males and females and tud progeny males and females at the early stages of metamorphosis (Figure ), suggesting they also play a non-sex-specific role in pupal somatic tissues. Several genes encoding products annotated as functioning in the female germline (found in Cluster 21) peak in transcript abundance in male and female somatic tissues at 48 hour APF (Figure ). However, by the later stages of metamorphosis, the levels of these transcripts remain high only in wild type females and drop to trough levels in wild type males, and tud male and female progeny.
The chromosomal distribution of genes with sex-biased expression in the male and female germlines was additionally analyzed. Genes expressed in the pupal male germline are underrepresented on the X chromosome and overrepresented on the left arm of the second chromosome (P
= 3.5 × 10-7
and 0.0072, respectively, hypergeometric test), both of which have been shown for genes expressed in adult male germline tissues [14
]. Interestingly, genes expressed in the pupal male germline are also overrepresented on the right arm of the third chromosome (P
= 0.024, hypergeometric test).
Global transcriptional profiles during metamorphosis
Hierarchical clustering was performed using all the data from each microarray experiment from the time course study, rather than using the data from each gene, to determine how similar global expression patterns are between males and females. When the tud progeny expression data was analyzed, the global expression profiles of males and females from each pupal time point were most similar to each other (Figure ). This was expected because very few genes with somatic, sex-differential expression were identified (see above). A clear distinction between overall gene expression at early stages (0–48 hour APF pupae) and late stages (72–96 hour APF pupae) was observed (Figure ). This is consistent with our cluster analyses (Figure ), where many genes appear co-regulated at either early or late stages of metamorphosis, but not at both early and late stages: 1,745 genes shared either peaks or troughs of transcript levels at multiple early stages (0–48 hour APF) or late stages (72–96 hour APF), while only 513 genes shared peaks or troughs of transcript levels at an both an early and a late time point.
Figure 5 Cluster of global expression profiles for Drosophila transcripts across metamorphosis. Dendrogram shows the similarity across transcriptional profiles at five time points during metamorphosis (0, 24, 48, 72, and 96 hour APF) for (A) male tud progeny (blue) (more ...)
When the wild type expression data and the tud progeny expression data were analyzed together, the clear distinction between early and late metamorphosis remained, as with the tud progeny expression data alone (Figure ). As expected, male-germline gene expression has a large effect on how the global transcriptional profiles cluster, with wild type males expression data always clustering separately from wild type females and from the male and female tud progeny. This effect appears to be less substantial at 0 hour APF, as the global expression profile of wild type males clusters closest to the data from the other three genotypes, suggesting that at the start of metamorphosis, many genes expressed in the male germline are not as abundant during early time points. The wild type female array experiments also cluster closely to, but separately from, the tud progeny array data, with the largest differences seen at 72 and 96 hour APF. This is consistent with gene expression in the female germline increasing at the end of metamorphosis (Figure ).
Sex hierarchy-regulated somatic sex-differential expression
Next, genes regulated by the sex hierarchy during pupal developmental stages were identified. Nearly all of the sexually dimorphic tissues are either patterned or undergoing morphogenesis to bring about the adult sexual dimorphisms during pupal stages. The 48 hour APF pupal stage was chosen because previous studies showed that FRUM
peaks at this stage [33
] and DSX shows high expression at this time [34
]. For these experiments, the array hybridizations were performed as direct comparisons using RNA from the two genotypes (see Table and Methods). Genes were first identified that had sex-biased transcript levels between wild type males and females by analyzing expression data from two different wild type strains, Canton S and Berlin (q
< 0.15), and from tud
progeny males and females (one-tailed t
< 0.15). This resulted in a set of 420 genes (320 and 100 genes with female- and male-biased expression, respectively; Additional file 10
). This is substantially more than was identified in the time course analysis for this time point (79 genes, see above); however this difference is likely due to the increased number of replicates (four replicates for each comparison versus three replicates in time course) and the decreased statistical error by directly comparing gene expression on the same array, as opposed to using a common reference RNA sample.
Given the larger number of somatic, sex-differentially expressed genes identified by this approach, it could be determined if there was a bias for chromosomal positions in these gene sets. Genes with somatic male-biased transcript levels at the adult stage are known to be underrepresented on the X-chromosome [14
]. There was not a similar bias for the 100 genes with somatic, male-biased transcript levels at the 48 hour APF pupal stage, but rather there was an equal distribution across all chromosomes (hypergeometric test, P
> 0.05). Interestingly, the genes with somatic female-biased transcript levels at this pupal stage were overrepresented with genes located on the X chromosome (90 genes, hypergeometric test, P
< 0.001). The previous study did not find any significant over- or underrepresentation on any chromosome for genes with adult somatic female-biased expression in the adult [14
Genes differentially expressed as a consequence tra
Next, genes were identified that were differentially expressed as a consequence of tra
, a gene in the sex hierarchy that encodes a pre-mRNA splicing factor required for the production of the sex-specific dsx
mRNA splice variants [35
]. Transcript levels in chromosomally XX flies mutant for tra
(hereafter called tra
pseudomales) were compared to wild type female flies. The tra
pseudomales produce DSXM
and look very similar to wild type males. Of the 420 genes that showed somatic sex-biased transcript levels, 95 genes were identified (72 female-biased and 23 male-biased) that are also significantly different between tra
pseudomales and wild type females (one-tailed t
< 0.15 for each test, Additional file 10
). In this experimental design genes were required to be differentially expressed in three different genotype comparisons of male and female gene expression (CS, Berlin, and tud
), precluding the identification of genes that are differentially expressed in only one strain. This is also true for the set of DSX-regulated genes identified below. As a validation of our experimental approach, Sxl
, and roX2
are all sex-differentially expressed in somatic tissues (wild type and tud
progeny comparisons). Only tra
is differentially expressed in the chromosomally XX, tra
pseudomale and wild type female comparison. This is expected as Sxl
, and roX2
are not regulated downstream of tra
in the sex determination hierarchy, but are regulated downstream of the primary determinate of sex, the X chromosome to autosome ratio (Figure ; [reviewed in [36
Of the 326 genes that are not regulated by TRA, a large portion (169) may be false negatives as their expression values are significant or close to significantly different (q < 0.30) in microarray experiments identifying genes regulated by dsx (see below). A gene regulated downstream of dsx should also be regulated downstream of tra. Another 19 have a q value close to the cutoff for significance in the tra microarray expression data (q < 0.30). Removing these 188 genes from consideration still leaves a large number of genes (138 genes) that are sex-differentially expressed independently of tra. A significant number of these 138 genes (45 genes; P < 0.05, hypergeometric test) are located on the X chromosome. It is possible that differences in transcript levels of these genes is due to differences downstream of Sxl, or X chromosome composition in males and females, suggesting that the dosage compensation process does not completely normalize expression between males and females for all genes on the X chromosome.
Genes differentially expressed as a consequence dsx
Next, gene expression between pupae that are transheterozygous for the dsxD
] – an allele that only produces the male-specific isoform (DSXM
) – and a dsx
null deletion allele (dsxm+r15
) was compared to gene expression in wild type females. These chromosomally XX, dsxD
pseudomales look very similar to wild type males, as they only produce DSXM
. Of the 95 genes that are sex-differentially expressed in somatic tissues and downstream of tra
, 66 genes were identified as being regulated downstream of dsx
< .015). Forty-six and 20 genes are more highly expressed in females and males, respectively, downstream of tra
(Table and Additional file 10
Genes expressed downstream of DSX at 48 hour APF.
Aside from the tra
gene itself, 28 genes were differentially regulated by TRA, but were not differentially expressed between dsxD
and wild type females. If genes that are close to the significance level (q
< 0.30; 6 genes) and genes with expression data in only one or two dsxD
comparisons (3 genes) are removed, 19 genes remain that are downstream of tra
, but not dsx
. Interestingly, none of these genes are significantly differentially expressed in similar experiments examining FRUM
regulation at this stage (data not shown). This suggests either an alternate branch of the sex-hierarchy downstream of tra
, possibly through dissatisfaction
], or the possibility of additional genes on which TRA acts to sex-specifically splice their pre-mRNAs, leading to differential abundance of transcripts due to differences in mRNA stability.
Requiring a gene to show statistical differences in expression in all direct microarray experiments yields a high confidence set of true positives regulated downstream of dsx
, but will likely generate false negatives. To identify additional dsx
regulated genes that might have been missed because of the stringency of having to pass multiple tests, genes were included that showed sex-differential, somatic expression, but which were not differentially expressed in the tra
microarray comparisons. These genes were required to be significantly differentially expressed in the dsxD
comparisons at a more stringent level (q
< 0.05) to avoid false positives. This yielded an additional 107 genes, with 75 and 32 showing female- and male-biased expression, respectively (Table and Additional file 10
). This study thus identified 173 genes regulated as a consequence of dsx
(DSX set; 121 and 52 genes with female- and male-biased expression, respectively).
Several of the genes with male- and female-biased expression in the DSX set with the highest fold change include those with products that might be involved in epithelial morphogenesis, imaginal disc morphogenesis or cuticle formation, based on their sequence identity. The 52 genes with male-biased expression contains seven such genes, including ecdysone inducible ImpE1 (FC = 4.8), miniature (FC = 3.8), and dusky-like (FC = 3.1). Among the 15 genes with the highest female-biased expression, four encode proteins with cuticular domains (Cuticular protein 97Eb, 50Ca, 97Ea, and 51A; FC = 5.9, 3.5, 3.3, and 2.8, respectively), as well as obstructor-A (FC = 2.5) and abdominal A (FC = 1.6). While it has long been recognized that cuticle deposition is tied to tissue morphogenesis and both are developmental events occurring during the middle of metamorphosis, the identification of several genes likely involved in sex-specific aspects of this process had not been determined until this study.
Six genes with female-biased expression regulated downstream of DSX at 48 hour APF have products with functions in the muscle or muscle differentiation (flightin, Limpet
, Tropomyosin 1
and Sarcoplasmic calcium-binding protein
]). This suggests that aspects of pupal muscle development occur in temporally distinct manner between males and females, and that this differential timing is regulated by DSX. It is not clear is if this is due to the development of sex-specific muscles or due to differences in the developmental rate of non-sex-specific muscles between males and females.
Characterization of the modes of DSX regulation
In our previous microarray study examining modes of DSX-regulated gene expression at the adult stage in head tissues, a large number of sex-biased genes were found that were either activated or repressed as consequence of dsx
activity in both males and females, but the extent of activation or repression was sex-specific [43
]. This mode of regulation was distinct from the previous descriptions of DSX-regulated gene expression based on the only known direct targets of DSX, Yolk protein 1
) and Yolk protein 2
activates Yp1 and Yp2
expression in the female fat body and DSXM
represses Yp1 and Yp2
expression in the male fat body tissues [45
To test if the set of genes regulated as a consequence of DSX activity contains genes that may be directly regulated by DSX, it was determined if the known DSX binding site sequences are present in the regulatory region of these genes [46
]. We searched for the presence of two DSX binding sites in the DNA sequence 2000 base pairs upstream from the transcription start and within the first intron, for each gene in our list (see Methods for search criterion) [45
]. Two DSX-binding sites were identified in 46 of the 165 (28%) DSX-regulated genes, a statistical overrepresentation as compared to all genes in the genome (P
= 0.0002, hypergeometric test), suggesting that a fraction of the genes identified here may indeed be direct targets of DSX. We note that in this study regulation by DSX may be direct or indirect.
To determine the modes of regulation by DSX in pupal stages, gene expression was compared between chromosomally XX and XY dsx
null flies and wild type females and males, respectively (hereafter called dsx
null comparisons). Data was examined for the 173 genes we identified here as being downstream of dsx
(DSX set; Table and Additional file 10
). Fifteen genes did not have enough expression data for statistical analysis or were not significantly differential expressed in either dsx
null comparison and were therefore not considered for further analysis. Of the remaining 158 genes, 151 show significant differential expression (q
< 0.15) in both dsx
null comparisons, which suggests regulation downstream of both DSXF
activity. The remaining seven genes (2 and 5 male- and female-biased genes, respectively) only showed significant differential expression in one of the dsx
null comparisons; these seven genes may possibly be regulated downstream of one isoform of DSX, a method of DSX regulation that was previously proposed for some genes with sex-differential expression in the adult [43
Of the 151 genes regulated as a consequence of dsx
in both sexes, 104 of the genes had female-biased expression and were more highly expressed in wild type females and males as compared to dsx
null females and males, respectively. This suggests that these genes are activated downstream of DSX in both females and males, but that DSXF
activity results in more potent activation. Forty of the 151 genes were male-biased and more highly expressed in male and female dsx
null flies than in wild type males and females, suggesting these genes are repressed as a consequence of DSX activity in both males and females, but DSXF
activity results in more potent repression. Thus, the majority of genes that are regulated as a consequence of dsx
are not regulated in the Yp
-like mode of regulation, but rather are regulated similarly in both sexes, with gene expression downstream of one isoform resulting in more potent activation or repression, as previously described in our studies of adult head tissues [44
]. Interestingly, Yp
-like regulation was observed for only seven genes in our pupal dataset: the genes with male-biased expression CG8086
and the genes with female-biased expression abdominal-A
, and CG9485
. It is possible that the Yp
-like mode of regulation may be the more common method of regulation for a particular class of genes (i.e., direct targets) or might be revealed to be the primary mode of dsx
regulation when higher resolution analyses are performed.
, which appears to be activated downstream of DSXF
in females and repressed downstream of DSXM
in males, is a well-characterized homeotic selector gene that was shown to be important for specifying segment identity [47
]. In the time course analyses above, abdA
was found to have female biased expression at 0, 24, 48, and 72 hour APF, with the highest expression difference between the sexes at 48 and 72 hour APF. Previous research examining 40–45 hour APF, suggested that ABD-A and DSX, along with Abdominal-B, act to regulate the expression level of a downstream target, bric-a-brac
, and lead to differential abdominal pigmentation between males and females [49
]. In that study it was shown that abdA
transcript levels in the abdominal epidermis do not vary between dsx
null animals and wild type animals, thus suggesting that abdA
is not regulated by DSX in this tissue. The dsx
-dependent differential expression of abdA
that we observed could be due to expression in other tissues, since here whole pupae were analyzed. Indeed, abdA
has been shown to be expressed and functional in several distinct tissues and cell types, including abdominal neuroblasts and the female genital disc [50
The proposed modes of regulation were validated by additional microarray experiments in which the male and female isoforms of DSX were over-expressed (data not shown). Of the 158 genes in the DSX-regulated set for which DSX modes of regulation was examined, 47 did not show significant differential expression in the experiments when we either over-expressed DSXF in females or DSXM in males. Of the remaining 111 genes, 35 were male-biased; these genes showed decreased expression when DSX was over-expressed, either in one or both of the DSX isoform over-expression experiments. Similarly, of the remaining 76 female biased genes, 74 showed increased expression levels when DSX was over-expressed, either in one or both of the DSX isoform over-expression experiments. Only two genes with female-biased expression (CG4484 and CG3244) showed decreased expression levels when DSXF was over expressed in females compared to control females, opposite of the predicted effect from our model.