Expression analysis of the speech-related genes FoxP1 and FoxP2 and their relation to singing behavior in two songbird species

Abstract

Humans and songbirds are among the rare animal groups that exhibit socially learned vocalizations: speech and song, respectively. These vocal-learning capacities share a reliance on audition and cortico-basal ganglia circuitry, as well as neurogenetic mechanisms. Notably, the transcription factors Forkhead box proteins 1 and 2 (FoxP1, FoxP2) exhibit similar expression patterns in the cortex and basal ganglia of humans and the zebra finch species of songbird, among other brain regions. Mutations in either gene are associated with language disorders in humans. Experimental knock-down of FoxP2 in the basal ganglia song control region Area X during song development leads to imprecise copying of tutor songs. Moreover, FoxP2 levels decrease naturally within Area X when zebra finches sing. Here, we examined neural expression patterns of FoxP1 and FoxP2 mRNA in adult Bengalese finches, a songbird species whose songs exhibit greater sequence complexity and increased reliance on audition for maintaining their quality. We found that FoxP1 and FoxP2 expression in Bengalese finches is similar to that in zebra finches, including strong mRNA signals for both factors in multiple song control nuclei and enhancement of FoxP1 in these regions relative to surrounding brain tissue. As with zebra finches, when Bengalese finches sing, FoxP2 is behaviorally downregulated within basal ganglia Area X over a similar time course, and expression negatively correlates with the amount of singing. This study confirms that in multiple songbird species, FoxP1 expression highlights song control regions, and regulation of FoxP2 is associated with motor control of song.

Keywords: Bengalese finch; basal ganglia; birdsong; variability; zebra finch.

Fig. 1.

Timelines for the behavioral groups used in this study. (A) Experimental design for time-course analysis of FoxP1 and FoxP2 behavioral regulation. On the day of the experiment, female birds remained alone in sound attenuation chambers for 2 h (green bar). NS males were discouraged from singing by the experimenter sitting nearby for 2 h (black bar). UD males sang alone in the isolation chamber for variable periods of time (red bars). Arrows indicate the time points at which birds were killed. (B) Experimental design for song variability analysis. Songs sung after the 2 h time point were analyzed for song variability. Birds were not killed in this experiment.

Fig. 2.

Representative exemplars of zebra and Bengalese finch song. (A) Spectrograms from a male zebra finch (ZF, top) and a male Bengalese finch (BF, middle) are shown. The red bar underneath each spectrogram indicates the length of one motif. Spectral derivatives of these motifs are shown underneath each spectrogram. (B) Markov chains generated from zebra finch and Bengalese finch songs. Letters denote syllables. Arrows represent the probability of syllable transitions. Thicker arrows indicate greater probabilities.

Fig. 3.

Representative brightfield photomicrographs of FoxP1 and FoxP2 mRNA expression patterns in a series of sagittal sections from one 2 h NS (left) and one 2 h UD (right) adult male Bengalese finch brain. Both medial and lateral sections are shown to enable display of the song control nuclei investigated here. (A) Nissl-stained sagittal sections. Locations of medial and lateral sections correspond to the level of sagittal plates 6 and 11, respectively, in the zebra finch atlas of Nixdorf-Bergweiler and Bischof (Nixdorf-Bergweiler and Bischof, 2007). (B) Schematic drawings based on the Nissl stains. A, arcopallium; Bas, basorostral pallial nucleus; HA, apical part of the hyperpallium; HD, densocellular part of the hyperpallium; LMAN, lateral magnocellular nucleus of anterior nidopallium; M, mesopallium; N, nidopallium; NC, caudal nidopallium; RA, robust nucleus of arcopallium; StL, lateral striatum; StM, medial striatum. (C) FoxP1 mRNA signals. (D) FoxP2 mRNA signals. Medial sections in A, C and D were adjacent or near adjacent to one another; similarly, lateral sections were adjacent or near adjacent. D, dorsal; C, caudal.

Fig. 4.

Representative brightfield photomicrographs of FoxP1 and FoxP2 mRNA expression patterns in a pair of sagittal sections from adult female Bengalese finch brain. Locations of medial and lateral sections correspond to the level of sagittal plates 6 and 11, respectively, in the zebra finch atlas of Nixdorf-Bergweiler and Bischof (Nixdorf-Bergweiler and Bischof, 2007). The medial plate shows the HVC and LMAN, and the lateral plate shows the HVC and RA in corresponding sections from male birds (Fig. 3).

Fig. 5.

FoxP1 mRNA expression in Area X of adult male Bengalese finches. (A) Brightfield photomicrograph of Nissl-stained hemi-coronal section with schematic drawing highlights song nuclei LMAN and Area X. Abbreviations as in Fig. 3B. (B) Representative images of FoxP1 mRNA expression at the level of Area X in 2 h NS (left) and 2 h UD (right) adult male Bengalese finch brain. There is no apparent effect of singing on expression levels. Location of sections corresponds to the level of transverse plate 11 in the zebra finch atlas of Nixdorf-Bergweiler and Bischof (Nixdorf-Bergweiler and Bischof, 2007). D, dorsal; L, lateral.

Fig. 6.

FoxP2 mRNA expression within Area X diminishes after birds sing undirected songs. (A) Top: schematic drawing based on a Nissl-stained hemi-coronal section shown with a control hemi-section incubated with sense RNA. Abbreviations as in Fig. 3B. Bottom: representative brightfield photomicrographs of FoxP2 mRNA expression patterns in hemi-coronal sections from Bengalese finches (left) and zebra finches (right) of different behavioral groups shown with corresponding Nissl-stained hemi-sections. D, dorsal; L, lateral. (B) Quantitative results of FoxP2 mRNA expression level within Area X relative to the ventral striato-pallidum (VSP). Boxes indicate s.e.m., points in boxes indicate means and whiskers indicate maximum and minimum values (2 h NS BF: N=7; 1 h UD BF: N=3; 1.5 h UD BF: N=6; 2 h UD BF: N=6; 2 h NS ZF: N=5; 1 h UD ZF: N=4; 1.5 h UD ZF: N=5; 2 h UD ZF: N=6; Kruskal–Wallis nonparametric ANOVA, ***P<0.001).

Fig. 7.

Correlation between FoxP2 and amount of singing. (A) In zebra finches, FoxP2 levels decrease as the amount of singing increases (P<0.0002). (B) FoxP2 levels also decrease as the amount of singing increases in Bengalese finches (P<0.0003). There is no significant difference between the two regression lines (P>0.05). The dotted rectangle indicates data from those birds that sang similar amounts of song for each species (see Discussion).

Fig. 8.

Behavioral changes in syllable self-identity. (A) Paired plot of syllable cluster self-identity in NS-UD and UD-UD conditions. The UD-UD condition had higher mean self-identity (*P=0.034, two-tailed paired bootstrap). (B) Representative spectral derivatives of five syllables from one cluster in the NS-UD and UD-UD conditions with self-identity scores reported.