Sexually dimorphic and developmentally regulated expression of tubulin-specific chaperone protein A in the LMAN of zebra finches

Abstract

Sex differences in brain and behavior exist across vertebrates, but the molecular factors regulating their development are largely unknown. Songbirds exhibit substantial sexual dimorphisms. In zebra finches, only males sing, and the brain areas regulating song learning and production are much larger in males. Recent data suggest that sex chromosome genes (males ZZ; females ZW) may play roles in sexual differentiation. The present studies tested the hypothesis that a Z-gene, tubulin-specific chaperone protein A (TBCA), contributes to sexual differentiation of the song system. This taxonomically conserved gene is integral to microtubule synthesis, and within the song system, its mRNA is specifically increased in males compared to females in the lateral magnocellular nucleus of the anterior nidopallium (LMAN), a region critical for song learning and plasticity. Using in situ hybridization, Western blot analysis, and immunohistochemistry, we observed effects of both age and sex on TBCA mRNA and protein expression. The transcript is increased in males compared to females at three juvenile ages, but not in adults. TBCA protein, both the number of immunoreactive cells and relative concentration in LMAN, is diminished in adults compared to juveniles. The latter was also increased in males compared to females at post-hatching day 25. With double-label immunofluorescence and retrograde tract tracing, we also document that the majority of TBCA+ cells in LMAN are neurons, and that they include robust nucleus of the arcopallium-projecting cells. These results indicate that TBCA is both temporally and spatially primed to facilitate the development of a sexually dimorphic neural pathway critical for song.

Keywords: ANOVA; HRP; HVC; LMAN; NGS; PBS; RA; TBCA; TRITC; analysis of variance; high vocal center; horseradish peroxidase; lateral magnocellular nucleus of the anterior nidopallium; neural development; normal goat serum; phosphate-buffered saline; robust nucleus of the arcopallium; sex chromosome; sex difference; song system; tetramethyl rhodamine isothiocyanate; tubulin-specific chaperone protein A.