We detected 2419 spots on the array that showed significant effects of sex (FDR p < 0.05), half of them (49%) with increased expression in males compared to females. However, of the targets with average effect sizes of 1.5-fold or greater, 300 were increased in males and 51 increased in females. A very large number (16,497) of the cDNAs exhibited a significant effect of age. This result is not particularly surprising, as the four ages investigated span the entire period of maturation for these birds, thus countless structural and functional changes in the brain would be expected. A relatively small number of cDNAs expressed a significant sex × age interaction, just 114 of the more than 20,000 spots on the array. These will be pursued in the future.
As our present goal was to identify genes involved in any aspect of masculinization of the song system, we chose to follow up on a set of 20 with increased expression in males compared to females across all ages. Most of these genes were initially selected based on substantially greater expression in males compared to females and location of the orthologous sequence in chicken on the Z chromosome (which was the only information available for birds at the time). In a few cases, genes were chosen for further analysis due to a high degree of sexual dimorphism in the absence of additional information. Because some gene identities have not been established (see above), we use GenBank accession numbers here to label genes.
Of the 20 cDNAs chosen for further analysis, 12 showed male-biased expression via qPCR (Table ). Two of these, with GenBank accession numbers DV946640 and CK306803, represented cDNAs from the same gene, sorting nexin 2 (SNX 2). Therefore, DV946640 was excluded from further analyses. GAPDH expression never differed significantly between the sexes.
Sexually Dimorphic Expression in Day 25 Zebra Finches Detected with cDNA Microarrays and Real-time qPCR
In situ Hybridization
Of the 11 cDNAs carried forward in the analysis, expression of 8 was detected in one or more song nuclei. Labeling was also detected in restricted regions outside of the song system, but was not quantified in the present study. The remaining 3 [GenBank: CK310754, DV947064, and DV948036] showed no specific staining in song control regions; they will not be discussed further.
Specific expression of CK303566 was detected in lMAN, Area X and RA, but not HVC (Figure ). The density of labeling was greater in males than females in all three areas (lMAN: t = 3.32, p = 0.008; Area X: t = 3.35, p = 0.007; RA: t = 3.89, p = 0.003; Table ).
Figure 2 Darkfield images from in situ hybridization depicting sexually dimorphic mRNA expression for CK303566in the zebra finch song system at day 25 post-hatching. Arrows delineate borders of song regions. This gene showed the most extensive sex differences (more ...)
Summary of Sexually Dimorphic Expression of Eight Putative Genes at Post-hatching Day 25
CK310795 (Methylcrotonyl-CoA carboxylase beta chain) showed specific expression in two song nuclei – area X and lMAN. In both cases, it was increased in males compared to females (Area X: t = 3.56, p = 0.005; lMAN: t = 2.92, p = 0.015; Figure ).
Figure 3 Darkfield images from in situ hybridization depicting sexually dimorphic mRNA expression for CK310795(Methyl-crotonyl carboxylase CoA) in the zebra finch song system at day 25 post-hatching. Arrows delineate borders of song regions. In lMAN (lateral magnocellular (more ...)
DV956689 was also expressed in area X and lMAN, but not in HVC or RA. Males showed significantly higher levels of expression than did females in both areas (area X: t = 3.85, p = 0.004; lMAN: t = 3.86, p = 0.004).
Three of the four song nuclei (lMAN, Area X, and HVC) exhibited specific labeling indicating SNX 2 mRNA [GenBank: CK306803]. It was increased in males in area X and HVC (t = 2.85, p = 0.017; t = 11.10, p < 0.001, respectively; Figure ). Expression between the sexes, however, did not differ in lMAN (t = 0.94, p = 0.370).
Figure 4 Darkfield images from in situ hybridization depicting sexually dimorphic mRNA expression for CK306803(Sorting nexin 2) in the zebra finch song system at day 25 post-hatching. Arrows delineate borders of song regions. In area X (or the portion of the medial (more ...)
CK313884 (17-beta-hydroxysteroid dehydrogenase type IV) showed enhanced expression compared to surrounding tissue in lMAN, area X, and HVC, but not in RA. Males had higher levels of expression in HVC than did females (t = 3.13, p = 0.008; Figure ). The sexes did not differ in the other two areas (lMAN: t = 1.63, p = 0.135; area X: t = 1.55, p = 0.150).
Figure 5 Darkfield images from in situ hybridization depicting sexually dimorphic mRNA expression for CK313884(17-beta-hydroxysteroid dehydrogenase type IV) in HVC at day 25 post-hatching. Arrows delineate borders of song region. Males (A) showed higher levels (more ...)
CK308959 mRNA appeared specific to lMAN and HVC. In HVC, it was increased in males compared to females (t = 3.38, p = 0.007). Males also showed somewhat higher levels of expression than did females in lMAN, though this difference was not statistically significant (t = 2.25, p = 0.048).
Only one area, lMAN, showed specific labeling for CK306648, with the intensity substantially greater than in surrounding tissue. It, however, did not significantly differ between the sexes (t = 0.51, p = 0.619). Results for CK303187 were the same (t = 0.47, p = 0.651).
Southern Blot Analyses
The corrected optical density representing each of the six genes with sexually dimorphic expression in the song system was significantly higher in males compared to females (all t > 3.464, all p < 0.004; data not shown). In parallel, each of the sequences represented in Table shared substantial identity with portions of the zebra finch Z-chromosome (2008 map, not yet annotated).