FoxP2 expression in avian vocal learners and non-learners

Abstract

Most vertebrates communicate acoustically, but few, among them humans, dolphins and whales, bats, and three orders of birds, learn this trait. FOXP2 is the first gene linked to human speech and has been the target of positive selection during recent primate evolution. To test whether the expression pattern of FOXP2 is consistent with a role in learned vocal communication, we cloned zebra finch FoxP2 and its close relative FoxP1 and compared mRNA and protein distribution in developing and adult brains of a variety of avian vocal learners and non-learners, and a crocodile. We found that the protein sequence of zebra finch FoxP2 is 98% identical with mouse and human FOXP2. In the avian and crocodilian forebrain, FoxP2 was expressed predominantly in the striatum, a basal ganglia brain region affected in patients with FOXP2 mutations. Strikingly, in zebra finches, the striatal nucleus Area X, necessary for vocal learning, expressed more FoxP2 than the surrounding tissue at post-hatch days 35 and 50, when vocal learning occurs. In adult canaries, FoxP2 expression in Area X differed seasonally; more FoxP2 expression was associated with times when song becomes unstable. In adult chickadees, strawberry finches, song sparrows, and Bengalese finches, Area X expressed FoxP2 to different degrees. Non-telencephalic regions in both vocal learning and non-learning birds, and in crocodiles, were less variable in expression and comparable with regions that express FOXP2 in human and rodent brains. We conclude that differential expression of FoxP2 in avian vocal learners might be associated with vocal plasticity.

Figure 1.

Identification of the zebra finch FoxP2 (zfFoxP2) mRNA. A, Schematic representation of the zfFoxP2 mRNA structure and its four predicted protein isoforms (I–IV). Positions of start (atg) and stop (tga) codons, the polyglutamine tract (polyQ), zinc finger (Zn-finger), and forkhead box (Fox) DNA-binding domains are shown. Two mRNA segments (splice1 and splice2) are subject to alternative splicing. The presence (+) or absence (–) of splice1 and splice2 leads to variation in the length of ORFs. Splice1 contains a stop codon that shifts the frame so that the ORF begins at the second atg, splice2 inserts 60 bp in-frame into the coding region. The four predicted protein isoforms are shown. For the calculation of their theoretical molecular weight, we used Peptide Mass (http://www.expasy.org/tools/peptide-mass.html). B, Summary of length [in base pairs (bp) and amino acid (AA)] of the zfFoxP2 isoforms (I–IV) and the length of the RT-PCR products spanning the alternatively spliced region. C, RT-PCR on RNA of different zebra finch tissues spanning the alternatively spliced region, but not the entire ORF, yields DNA fragments of the expected sizes shown in B. D, Northern blot analysis of 20 μg of total RNA from adult zebra finch brain and lung was performed with a ³²P-labeled DNA fragment spanning bp 114–959 (relative to the first start codon of isoform III). Ethidium bromide staining of 18S and 28S ribosomal bands demonstrates equal RNA loading. The different zfFoxP2 transcripts are indicated with arrows. E, Western blot analysis of 50 μg of brain nuclear protein extract from a 40-d-old male zebra finch reveals a zfFoxP2 protein corresponding in size to either isoform I or II, recognized by a polyclonal antibody raised against aa 613–715 of mouse Foxp2 (Lu et al., 2002).

Figure 2.

Embryonic FoxP2 mRNA (A–C) and protein (D–F) expression. Sagittal sections through stage 26 (A) and 34 (B) zebra finch embryos show expression in presumptive striatum (arrowheads) and presumptive dorsal thalamus (arrow). The heads face toward the right. C, Embryonic chicken brain (embryonic day 13) had strong expression in the developing striatum and also in the pallial and subpallial germinal ventricular zone, shown in a frontal right hemisection. The FoxP2 mRNA label appears white in dark-field illumination in A–C. D–F, FoxP2 expression in a stage 26 zebra finch embryo frontal sections. FoxP2 immunoreactivity is brown, and cresyl violet-stained cells are purple/blue. D, A prominent band of FoxP2-positive cells is visible among cresyl violet-stained neurons in the ventrolateral telencephalic vesicle. E, The floor plate at the rostral end of the mesencephalic vesicle (arrowhead) has many FoxP2-expressing cells that seem to disperse laterally (arrows). F, At limb levels of the spinal cord, floor plate neurons expressed FoxP2 (arrowhead), as did a population of neurons in ventral cord (arrows). Scale bars: A–C, 2 mm; D–F, 100 μm.

Figure 3.

Differential FoxP2 expression in Area X during post-hatch zebra finch development (A–F). Area X expressed more zfFoxP2 than the surrounding striatum only at PHDs 35 and 50 (C, D, arrowheads), which is the time when zebra finches learn to imitate song. G and H show the results of autoradiographic densitometric quantification of expression levels at the different ages (n = 3 for each age). The ratio of expression between Area X and the surrounding striatum increased during the phase when song imitation occurs on PHDs 35 and 50 (G). Absolute levels of FoxP2 expression in the nidopallium did not change throughout development, whereas in the striatum (outside of Area X) they decreased slightly from PHDs 15 to 25 and reached adult levels by PHD 35 (H). Scale bar (in A): A–F, 2 mm.

Figure 4.

Different adult vocal learners (A–G), non-learners (H), and a crocodile (I) shared the FoxP2 expression pattern in the striatum and dorsal thalamus (DT) but differed in expression levels in the striatal vocal nucleus (Area X/VAS/MMSt). Area X of chickadees (sampled in the fall), strawberry finches (sampled on long day photoperiod), and canaries (sampled in July) expressed more FoxP2 in Area X than in the surrounding striatum (A–C), reflected in higher expression ratios (bars A–C in J). Area X of song sparrows (sampled in spring) expressed slightly less FoxP2 than the surrounding striatum (D; bar D in J), as did Bengalese finch (E; bar E in J). The rufous-breasted hermit hummingbird (F) had slightly higher expression in the VAS, and the parrot (G) did not show a difference between vocal nucleus MMSt and the surrounding striatum. The adult ringdove (H), a bird that does not exhibit vocal learning and lacks telencephalic vocal nuclei, expressed high levels of FoxP2 mRNA in the striatum and DT, as did a crocodile (I). The arrow in C points to the high levels of FoxP2 expression in the substantia nigra pars compacta. M, Mesopallium; MO, oval nucleus of the mesopallium; N, nidopallium; St, striatum; VAS, vocal nucleus of the anterior striatum; MMSt, magnocellular nucleus of the medial striatum. Scale bars (in A for A–E;in H for H, I), 2 mm.

Figure 5.

FoxP2 expression in Area X of adult canaries varied seasonally. Area X expressed noticeably more FoxP2 than the surrounding striatum only during the months of July, August, and September, resulting in higher ratios of Area X to striatum expression (the bar graph shows the mean ratios for each month, and superimposed points represent the values for individual birds).

Figure 6.

FoxP2 expression in distinct populations of neurons in adult zebra finches. Low (A) and high (B) magnification of a sagittal section showing the dorsolateral extent of the subpallial–pallial (P) border with the striatum (St; black dashed line), where clusters of cells in the dorsal and lateral striatum express FoxP2 (arrowheads; brown immunoreactivity). Dorsal is up, and rostral is to the right. C, These clusters (arrowheads; black-brown immunoreactivity) are characterized by dense ChAT fiber staining (lighter brown immunoreactivity). D, Clusters visualized with cresyl violet stain. E, FoxP2-immunoreactive cells within the clusters are neurons as shown by double labeling with fluorescent anti-Hu (red) and anti-FoxP2 (green). F, Higher magnification in the dorsal thalamus shows that the cytoplasmic neuronal anti-Hu antibody (red) colocalizes with nuclear FoxP2 antibody staining (green). FoxP2-negative nuclei can been seen in blue, stained with nuclear 4′,6-diamidino-2-phenylindole DNA stain. G, Some FoxP2-positive cells are recognized by anti-PSA-NCAM antibody, a cell adhesion protein (PSA-NCAM, red; FoxP2, green; TOPRO3 nuclei, blue). H, Striatal neurons also coexpress DARPP-32 (red) and FoxP2 (green) and appear to be innervated by TH-positive (red) terminals (I). Colabeling with neurochemical markers for three different striatal interneuron populations [ChAT (J), nNOS (K), or parvalbumin (L) (brown cytoplasmatic labeling; arrowheads)] revealed that FoxP2 (black nuclear labeling; arrows) was not expressed in these cell types. Scalebars: A, B, 100 μm; C–E, 50 μm; F–L, 10 μm.

Figure 7.

FoxP2 expression in subtelencephalic regions was associated more with afferent sensory or multimodal areas rather than with pure motor areas. Auditory nucleus MLd (dorsal part of the lateral mesencephalic nucleus) expressed FoxP2 (white dark-field label in A and brown label in B; both surrounded by yellow arrowheads). In contrast, the dorsomedial motor nucleus of the intercollicular region (DM), which controls vocalizations, showed little mRNA and immunoreactivity for FoxP2 (A, B, black arrowheads) but strong parvalbumin immunoreactivity (C) (Braun et al., 1985). Also, FoxP2-immunoreactive cells were seen in the visual thalamic nucleus rotundus (D), cerebellar Purkinje cells (E), specific layers of the optic tectum in the midbrain (F), and brainstem nucleus inferior olive (G) but not in the tracheosyringeal portion of the nucleus of the hypoglossal nerve nXIItx (I). We took advantage of the strong parvalbumin immunoreactivity of nXII to unambiguously identify this nucleus (adjacent section to I stained with parvalbumin in H) (Wild et al., 2001). Immunoreactivity in dark-field images appears white, and in bright-field photomicrographs brown. A, D, and E–G are sagittal sections, rostral is to the right, and B, C, H, and I are frontal sections. Dorsal is up in both orientations.

Figure 8.

Expression pattern of FoxP1 was distinct from but partially overlapping with that of FoxP2. A, FoxP1, like FoxP2, was expressed in the dorsal thalamus and striatum in adult zebra finches (A). In addition, it was expressed in vocal nuclei HVC, RA, and Area X (but not lMAN) at higher levels than their surrounding regions and in the mesopallium. Both male (B) and female (C) strawberry finches, male song sparrow (D), as well as the parrot (F) expressed more FoxP1 mRNA in Area X (MMSt in parrot) than in the surrounding striatum. E, A vocal non-learner, the ring dove, also expressed FoxP1 mRNA in the subpalllial and pallial areas. G, The crocodile had a telencephalic pattern very similar to that of birds. All sections are sagittal, except the parrot sections in F, which are frontal. Scale bars: A–D, 1 mm; E–G, 2 mm.