Birdsong "transcriptomics": neurochemical specializations of the oscine song system

Abstract

Background: Vocal learning is a rare and complex behavioral trait that serves as a basis for the acquisition of human spoken language. In songbirds, vocal learning and production depend on a set of specialized brain nuclei known as the song system.

Methodology/principal findings: Using high-throughput functional genomics we have identified approximately 200 novel molecular markers of adult zebra finch HVC, a key node of the song system. These markers clearly differentiate HVC from the general pallial region to which HVC belongs, and thus represent molecular specializations of this song nucleus. Bioinformatics analysis reveals that several major neuronal cell functions and specific biochemical pathways are the targets of transcriptional regulation in HVC, including: 1) cell-cell and cell-substrate interactions (e.g., cadherin/catenin-mediated adherens junctions, collagen-mediated focal adhesions, and semaphorin-neuropilin/plexin axon guidance pathways); 2) cell excitability (e.g., potassium channel subfamilies, cholinergic and serotonergic receptors, neuropeptides and neuropeptide receptors); 3) signal transduction (e.g., calcium regulatory proteins, regulators of G-protein-related signaling); 4) cell proliferation/death, migration and differentiation (e.g., TGF-beta/BMP and p53 pathways); and 5) regulation of gene expression (candidate retinoid and steroid targets, modulators of chromatin/nucleolar organization). The overall direction of regulation suggest that processes related to cell stability are enhanced, whereas proliferation, growth and plasticity are largely suppressed in adult HVC, consistent with the observation that song in this songbird species is mostly stable in adulthood.

Conclusions/significance: Our study represents one of the most comprehensive molecular genetic characterizations of a brain nucleus involved in a complex learned behavior in a vertebrate. The data indicate numerous targets for pharmacological and genetic manipulations of the song system, and provide novel insights into mechanisms that might play a role in the regulation of song behavior and/or vocal learning.

Figure 1

(A) Schematic of a male zebra finch brain showing the approximate locations of HVC and underlying auditory shelf, as well as other major nuclei of the song system and major pallial and subpallial brain divisions. HVC provides major input into: a direct motor pathway (black nuclei), and an anterior forebrain pathway (white nuclei). Laser capture microdissection (LCM) was used to conservatively sample HVC and the underlying auditory shelf (LCM sites denoted by dotted ovals). (B and C) Under brightfield (B, white arrows) HVC could be identified by a characteristic bump on the surface of the brain and the presence of large cells and cell clusters; under dark-field (C) mylenated fibers are seen close to the ventral boundary of HVC. LCM dissections were confirmed by examination of the section after LCM (D), as well as of tissue adhered to the capture cap (E). Abbreviations: A, anterior; P, Posterior; D, Dorsal; V, Ventral; DLM, medial dorsolateral thalamic nucleus; LMAN, lateral magnocellular nucleus of the anterior nidopallium; nXIIts, tracheo-syringeal portion of the hypoglossal nerve nucleus; RA, robust nucleus of the arcopallium; RAm, nucleus retroambigualis; X, striatal area X; Arco., arcopallium; Nido., nidopallum; Meso., mesopallium; Hyper., hyperpallium.

Figure 2. (A–D) Representative in situ hybridization autoradiograms of parasagittal sections of adult male zebra finches at the level of HVC (∼1.4 to 2.4 mm from the midline).

(A, left) Schematic depicting the major telencephalic song nuclei (in black), thalamorecipient areas (i.e. L2a, Bas), and major brain subdivisions. (A, center and right) Expression of known HVC markers. The other panels (B–D) depict representative autoradiograms of HVC markers that are exclusive to HVC (B), enriched in HVC and other song nuclei, such as LMAN (C, left), or area X (C, center and right; inset shows area X from a more medial section), enriched in 3/4 major song nuclei (D, left and middle), or are negative markers (D, right). Notice that some markers are also enriched in distinct pallial and striatal areas, and/or primary sensory areas. Scalebar: 1 mm. Gene abbreviations in Table S1. Anatomical abbreviations: A, Arcopallium; Bas, nucleus basorostralis; Cb, Cerebellum; GP, globus pallidus; Hp; Hippocampus; H, Hyperpallium; L2a, subfield L2a of field L; LMAN, lateral magnocellular nucleus of the anterior nidopallium; M, Mesopallium; N, Nidopallium; RA, robust nucleus of the arcopallium; Ot, optic tract; Rt, nucleus rotundus; St; striatum.

Figure 3

(A and B) Darkfield (A) and brightfield (B) views of nissl stained emulsion autoradiography depicting the expression of S100B (white signal) and CRHBP (black signal and arrowheads) in HVC, respectively; arrows delineate the ventricle. (D) High magnification bright-field views of emulsion autoradiography depicting examples of high expression (black grains) over the majority of large HVC cells (C; Arrows), a portion of these cells (D), or virtually all large and small cells (E) in sections hybridized with antisense probes directed against follistatin (FST), a serotonin receptor (5HT1F), and a microtubule associated protein 4 (MAP4), respectively. White arrow in C indicates an example of a non-labeled small cell. Scalebars: 100 µm in A and B; and 10 µm in C–E. See Fig. 2 legend for anatomical abbreviations.

Figure 4. AgBase (http://www.agbase.msstate.edu/) was used to assign GO terms (www.geneontology.org) to as many genes as possible from the FDR <0.05 marker list, and to a set of a randomly sampled 1415 ESTIMA Unigenes expressed in zebra finch brain (see text for details).

(A and B) Bar graphs show the percentages of genes that were categorized as involved in a specific biological process (A) or activity (B) that minimally describes 3% of the genes in ESTIMA (Grey columns) or in the FDR<0.05 marker lists (Black columns). For each category, black columns with higher or lower percentages than the corresponding grey column are considered over- and under-represented in HVC, respectively. Because some genes are represented by more than one category, percentages in both graphs sum to more than 100%.

Figure 5

(A–C) Bright-field high power Nissl stained views of NRP1 emulsion autoradiography at the level of HVC. Black arrowheads indicate examples of small cells with high levels of emulsion grains (A), white arrows point to larger neurons with no detectable expression (see also C). (D) Low power dark-field autoradiography for SEMA3A in a parasagittal section at the level of RA. Note intensely labeled cells that align along the lamina arcopallius dorsalis (LAD). (E) Camera lucida drawing of the ventral nidopallium and arcopallium from the section shown in D. Individual cells expressing SEMA3A are represented by solid circles. For anatomical abbreviations see text. Scalebars: 10 µm in A–C; 100 µM in D; 1 mm in E.

Figure 6. (A–F) Phosphorimage autoradiograms showing the patterns of expression for nicotinic (in A, B and E, F) and muscarinic (in C, D) acetylcholine receptor subunits in sections hybridized with antisense probes direct against various receptor subunits.

See text for additional details and gene name abbreviations. Arrows indicate the locations of nucleus HVC in each as identified under bright-field illumination in an adjacent Nissl stained section, arrowheads indicate the location of area X. Scale bar: 1 mm.

Figure 7. Networks describing the involvement of HVC markers in the formation of adherens junctions (A) and in the mediation of focal cell adhesions to the extracellular matrix (ECM) (B).

Gene abbreviations are in the text and Tables S1 and S3. Rectangles constitute central nodes with specific receptor and/or structural properties, genes in ovals play a primarily regulatory role. Arrows indicate a positive direct interaction, solid lines depict bi-directional protein-protein interactions. Not all connections and nodes are included; genes not required for network assembly, or not present on the arrays were omitted for clarity. Grey elements represent differentially expressed genes from our primary or secondary (asterisks), white symbols indicate genes that were either not differential (solid), or not on the array (dashed).

Figure 8. Gene abbreviations are in the text and Tables S1 and S3.

Rectangles constitute important nodes in the network that have receptor activities, genes presented in ovals play either a regulatory role, or are products of transcription (shown in the nucleus). Not all connections and nodes are included; genes not required for network assembly, or not present on the arrays were omitted for clarity. Rectangular and oval symbols depicted in grey indicate genes from our primary or secondary lists (asterisks), symbols or fractions of symbols shown in white were either not differentially expressed (solid) or not on our array (dashed). Major cellular compartments are indicated along the top or by the presence of phospholipid membranes.