Conceived and designed the experiments: PVL CVM. Performed the experiments: PVL KLR. Analyzed the data: PVL KLR CVM. Contributed reagents/materials/analysis tools: DFC. Wrote the paper: PVL CVM. Helped conceive the research plan and is the primary PI on of Songbird Neurogenomics initiative that generated and sequenced ESTs for the ESTIMA: DFC. Songbird clone collection, and printed cDNA microarrays: DFC.
The authors have declared that no competing interests exist.
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.
Using high-throughput functional genomics we have identified ∼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.
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.
The emergence of high-throughput functional genomics has made it possible to identify novel relationships between genes, brain, and behavior. Microarray platforms, in particular, have given enormous momentum to the study of brain gene regulation in the contexts of sensory/motor processing, learning, the formation of memories, aging, and the onset of diseases
Vocal learning is a rare trait, expressed in just three orders of birds (e.g. hummingbirds, parrots, songbirds), cetaceans, and humans, where it serves as a basis for the acquisition of spoken language. In vocal learning birds, the memorization and production of song share many important parallels with the process of speech acquisition in humans and depends on a set of telencephalic nuclei referred to collectively as the song control system
In songbirds, the caudo-dorsal portion of the nidopallium (a part of the avian pallium that is thought to share a common origin with mammalian cortical regions) encompasses both HVC, a specialized nucleus of the song system that is unique to songbirds, and the Shelf, a component of the central auditory system that is present in all birds (
(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;
HVC provides major inputs to both the direct pathway for vocal-motor control and the anterior forebrain pathway (
To identify markers of HVC we used microarray screening of laser capture microdissected samples from HVC and the adjacent Shelf region combined with bioinformatics for a comprehensive analysis of molecular genetic specializations of HVC. Our effort resulted in the identification of over 250 differentially expressed genes that are likely to be related to the unique properties of this song nucleus. Moreover, our bioinformatics analysis provide new perspectives on the roles that individual genes and genetic pathways might play in various aspects of cellular morphogenesis, neurogenesis, steroidogenesis, cellular excitability, neurotransmission, cell survival, steroid and retinoid sensitivity, and gene regulation in HVC.
Despite major progress towards understanding the anatomical and electrophysiological properties of song nucleus HVC, the nature of the molecular mechanisms and biochemical/genetic programs underlying these properties has remained largely unknown. To address this gap, we searched for differentially expressed genes in HVC versus the adjacent Shelf, an auditory area that is also part of the dorso-caudal nidopallium but not part of the song system (
To accurately and reproducibly obtain HVC and Shelf samples we used laser capture microscopy of frozen brain sections (
Using conservative ANOVA and protected post-hoc t-tests (Genespring) we identified 390 spots with significant differential expression (FDR<0.05) in HVC as compared to Shelf (
Differentially Expressed cDNAs (FDR = 0.05) | + | − | ND | Total |
Redundant (based on 5′ EST reads) | 17 | 30 | NA | 47 |
Non-redundant and unannotated (Unknowns) | 31 | 31 | NA | 62 |
Non-redundant and Annotated (Candidate Markers) | 130 | 151 | NA | 281 |
Present in Primary Gene list |
6 | 5 | NA | 11 |
Present in Secondary Gene lists |
4 | 6 | 3 | 13 |
Primary Gene list | 21 | 4 | NA | 25 |
Secondary Gene list | 1 | 1 | 3 | 5 |
ND = Not Differential.
NA = Not Applicable.
Previously identified Markers presented in
Primary List (FDR<0.05) presented in
Secondary Lists presented in
Two genes gave no in situ signal (see text for details).
Providing further validation for our screening, many known HVC markers were present on our primary (n = 11) and secondary (n = 13) lists (presented in
(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
To further validate our screening, we performed
To determine whether some of our HVC markers might be regulated by singing behavior or revealed by comparisons with different tissues, we cross-referenced our primary gene list against lists from two separate and unrelated microarray studies of HVC. We first compared our list to that derived from a comparison between HVC and the whole brain (
Our in situ expression analysis revealed that many HVC markers are also expressed in other song nuclei and brain subdivisions in various combinations (
Emulsion analysis further revealed that HVC markers have distinct cellular distributions. For example, S100B is expressed in a subset of cells that are uniformly distributed throughout HVC (
(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 (
We used GO analysis to identify biological processes and/or molecular functions that are over- or under-represented among HVC regulated genes (i.e., those differentially expressed between HVC and Shelf) as compared to a larger list representing a broader universe of genes expressed in the songbird brain (i.e. a subset of the ESTIMA collection of brain-expressed genes). Because GO comparisons do not take into account the magnitude or direction of gene regulation (up or down), all HVC markers were included in the analysis. Several GO categories were over-represented among HVC markers, including signal transduction, ion transport, and synaptic transmission (
(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%.
As presented next, we were able to classify the markers in our primary list into categories representing specific genetic, biochemical and cellular functions (
PARD3, PTPRF, RHOB, WASL | |
PARD3 | |
EPB41L2, PRKCD | |
COL21A1, COL4A2, DCN, FNTM2, |
|
ADAMTS8, CPNE2, CPNE8, PDGFRA, |
|
ADAM23, SCUBE1, |
|
NRP1, PDZRN3, PLXNA4 | |
PLXNA1, SEMA3A, |
|
DOK4, MCF2, NDRG4, S100B, STMN3, TNR | |
BMPR2, GAS7, LPPR4, PLD1, |
|
ANK1, |
|
MAP1B, MAP4, S100B, STMN3 | |
NEFH, NEFM, NEFL | |
none |
Tentative gene identifications are underlined (see
CHRNA5, CHRNA7, DIP2A, |
|
GLRA2, GRIA4, GRIK2, GRM1, |
|
CCK, MARCKS, TAC1 | |
CADPS2, |
|
DPP6, DPP10, KCNA1, ATP1B4 | |
Tentative gene identifications are underlined (see
none | |
CACNA1G, CACNG4 | |
ANXA6, CABP1, CADPS2, CAMK1D, PVALB, RCAN2, RGS12, S100B, |
|
CAMK2D, CPNE2, CPNE8, GPR98, HPCAL1, KCNIP1 | |
CAMK1D, MAPK11, MGC42105, PPP4R2, RP6-213H19.1, STK11IP | |
ARPP21, CAMK2D, LPPR4, PRKAR2B, PRKCD, PTPRZ1, SNRK | |
PLD1, PTER | |
GRM1, |
|
MCF2, |
|
ARL5B, DOCK4, RAB36, RASD2, RASGRF1, RASL11A, RASL12, RGS12, |
Tentative gene identifications are underlined (see
AIM1, GBAS, LMO2, LMO3, MCF2, MAP4, NETO1, QSOX1, RP6-213H19.1, SYF2, UBE2E1 | |
ENOX1, |
|
DCN, FST, NBL1, PDZRN3, SAP30L, THBS4, ZEB2 | |
BMPR2, CHST11, FAM5C, PRKCD, RSPO3, ZNF423 | |
No hits | |
NOP5/NOP58, PDGFRA, PRR5, RGS12 | |
GPC3, GPI, RGS12, ST8SIA4, TBR1, TNR | |
ARHGDIB, |
|
AIFM1, RP6-213H19.1, |
|
ARHGDIB, BAI3, FAM152A, PRKCD, RPRML, SCN3B, SHC1, SHC3, SNRK, TNFAIP8L3, |
Tentative gene identifications are underlined (see
ENDOGL1, PPP4R2, SAP30L | |
CNOT6, NAP1L4, NOL4, NOP5/NOP58, SNRK, |
|
CAMTA1, DIP2A, FOSL2, NDRG4, SOX6, SYF2, THOC4, TBR1 | |
PRKRIP1, RPL18A | |
PAIP1 | |
CRHBP, NR3C2 | |
none | |
RGS4 | |
EPB41L2, TMEPAI | |
C1orf34, QSOX6, SNCG, |
|
CACNA1G, CYP19A1, CYP1B1, PRKCD, RASL11A | |
none | |
− | ABCA1, DHRS2 |
ALDH1A2, ANXA6, LMO2, LMO3, NBL1, NDRG4, PLD1, RHOB, RP6-213H19.1, SACS, TNR | |
AQP1, CAMK2D, |
Tentative gene identifications are underlined (see
AHNAK2, COL4A2, NRP1, RCAN2 | |
ADAMTS8, ANTXR2, BAI3 | |
AHNAK2, ABCC5, CD59 | |
none | |
ADAM23, CPM, LGMN | |
ADAMTS8, MMEL1, |
Tentative gene identifications are underlined (see
Genes that have been linked to the regulation of cell structure, including cellular adhesion, neurite outgrowth and extension, and cytoskeletal organization represented nearly 25% of our primary list. Among these, we detected a small cluster involved in tight junctions and a larger cluster related to cadherin-catenins, which are major components of adherens junctions in neuronal and epithelial tissues. The latter included regulators of catenins (PTPRF, PPFIBP,
Several markers represented components of the extracellular matrix (ECM) or their receptors (e.g. integrins and receptor tyrosine kinases), known to be involved in the formation and maintenance of focal adhesions. This included the HVC-enriched collagens (COL21A1, COL4A2, COL2A1*, COL6A1*, COL10A1* and COL12A1*), decorin (DCN), tenascin (TNR), thrombospondin (THBS4), and a fibronectin III domain-containing protein (FNTM2), all of which are ECM components, as well as an alpha-integrin (
A significant cluster has been linked to axonal guidance. This included the upregulated neuropilin (NRP1) and plexin-A4 (PLXNA4), which together comprise the receptor for semaphorins 3A and 6A (SEMA3A and
(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.
Finally, we found several clusters containing genes implicated in various aspects of neurite outgrowth, extension, and in the initial stages of the formation of axonal processes, as well as cytoskeletal organization. For example, several genes involved in promoting (DOK4, MCF2, NDRG4) or inhibiting (STMN3, TNR and S100B) neurite extension/outgrowth were higher in HVC than in the Shelf, while the genes in this category that were lower in HVC are involved in promoting neurite outgrowth and/or dendritogenesis. Another large cluster consisted of markers implicated in cytoskeletal organization through interactions with actin (
Our microarray data confirmed the lower expression in HVC of several members of the ionotropic and metabotropic glutamate receptor family (GRIA4, GRIK2, GRM1,
Several cholinergic receptor subunits had high expression in HVC, including both nicotinic (CHRNA5, CHRNA7) and muscarinic (CHRM4*) family members, as well as PDZRN3, a ring finger protein implicated in acetylcholine receptor clustering. In contrast, other members of the cholinergic receptor family were not differential (CHRNA2, CHRNA9, a clone with similarity to both CHRNA2 and CHRNA4, and CHRM2) or not on the array (CHRNA1/3/6, CHRNA8, which is unique to birds, nicotinic beta-subunits CHRNB1/2/3, and CHRM1/3/5).
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.
We detected the HVC enrichment of several components related to serotonin-mediated signaling. While serotonin receptor subunits HTR1B and HTR7A were not differential,
We detected several markers related to peptidergic transmission, including the peptide secretory granule protein CHGB. In particular, the precursor of neurotensin/neuromedin N (NTS), a peptide whose release is mediated by
Several markers represented structural or regulatory components of sodium and potassium channels. Our analysis revealed that multiple K-channel subfamily members are often co-regulated at the exclusion of other members or subfamilies that would be predicted to confer similar properties. For example, among fast-inactivating subunits, KCNA1 and its accessory subunit KCNAB1* were higher in HVC (KCNA4 and KCNA6/7 were not differential and other members were not on the array). In contrast, KCND2 was lower in HVC, and two dipeptidyl-peptidases (DPP6 and DPP10) that can act as KCND-type inhibitory accessory subunits were enriched in HVC. Among delayed-rectifier subunits, KCNC1* and KCNC3* were up in HVC, whereas KCNC2 and its modulatory subunit (KCNIP1) were down. Although KCNB delayed rectifiers were not on the array, they also appeared to be down in HVC based on the relative lower expression of cooperative subunits (KCNF1, KCNG1*, KCNS2*), and an enrichment of an inhibitory subunit (KCNV1*) of this subfamily. Finally, two open-rectifying leak channels (
Several markers have been linked to calcium entry and binding, and the activation of signaling and phosphorylation cascades. We observed that calcium-channel related genes generally had lower expression in HVC, including a T-type low-voltage activated subunit (CACNA1G), an L-type channel (CACNA1D*), and an accessory subunit (CACNG4) known to associate with L-type channels. Other subclusters could be classified as calcium-dependent, capable of binding calcium, or calmodulin-like in their activity. Among these, we detected an HVC enrichment of genes involved in calcium-dependent liposome binding (ANXA6) and dense-core vesicle exocytosis (CADPS2;
Several markers were related to protein phosphorylation/dephosphorylation. Besides calcium-sensitive genes, these included a mitogen-activated protein kinase (MAPK11), serine/threonine kinases (MGC42105, PRKCD, PTPRZ1, RP6-213H19.1, SNRK), a cAMP-dependent protein kinase (PRKAR2B), protein phosphatases (ARPP21, LPPR4, PPP4R2), and phosphorylated proteins with phospholipase or phospho-(di/tri)-esterase activity (PLD1, PTER,
Nearly a quarter of the HVC markers were linked to cell proliferation, migration, differentiation and survival. Specifically, one large cluster is involved in cell proliferation and cycle progression, including HVC-enriched genes that mediate non-p53 tumor suppression (AIM1, GBAS, NETO1, UBE2E1; LMO2, LMO3), interact with cyclin D1 (SYF2), or generally relate to cell proliferation (QSCN6), as well as a variety of markers of low expression associated with tumor suppression, growth arrest and/or proliferation.
A second large cluster is related to the TGF-beta signaling pathway. Specifically, while a single component of the TGF-beta receptor (TGFBR2*) was enriched in HVC, several inhibitors of TGF (DCN, THSB4, LTBP1*) or activin (FST) signaling were also enriched. Several positive mediators of signaling had lower expression in HVC, including an activator of TGF-beta signaling (RSPO3) and an activin receptor component (ACVR1*; no activins were on the array). Other TGF-beta-related genes were not differential, including the only TGF-beta on the array (TGFB3), as well as a TGF-beta inducible nuclear protein (TINP1), a TGF-beta induced apoptosis protein (FAM130A2), an activin A receptor (ACVR2A), and downstream regulatory proteins (SMAD1, 2, 6 and 9). A subset of TGF-beta related genes belonged to the bone morphogenetic protein (BMP) pathway, including the HVC-enriched BMP receptor antagonists (NBL1, ZEB2), and a BMPR2 interactant (PDZRN3), as well as a BMP receptor (BMPR2) and two mediators of BMP activation (CHST11, and ZNF423), and the BMP target FAM5C, which were low in HVC. The only BMP on the array (GDF3) was not differential, the BMP signaling inhibitor NOG was highly expressed in HVC but not differential, and other BMP receptors (BMPR1A, BMPR1B and AMHR2) were not on the array. A smaller cluster of genes with lower expression in HVC included the platelet-derived growth factor receptor A (PDGFRA), regulators of the PDGF-pathway (PRR5 and RGS12), and PDGF-inducible NOP5/NOP58.
Other markers have been specifically linked to apoptosis and/or p53 tumor-suppressor function. Among HVC-enriched genes were an apoptosis-inducing factor (AIFM1, a.k.a. programmed cell death 8, or PDCD8; the PDCDs 2, 4, 5, and 6 were not differential), a kinase (RP6-213H19.1) that interacts with PDCD10, and a secreted glycoprotein (
Some HVC markers have been linked to cell migration, differentiation and survival, and play important roles in the maturation and integration of post-mitotic cells into mature circuitry, and patterning during development. These included a cell surface heparan sulfate proteoglycan (GPC3), a neurotrophic factor (GPI), and genes involved in the regulation of adhesion and ECM formation during neural development (ST8SIA4, TBR1, TNR), which were enriched in HVC, while known mediators of migration (
A number of HVC markers could be classified as being related broadly to gene expression regulation. For example, two histone acetylation/deacetylation proteins (PPP4R2, SAPL30), a chaperone complex protein related to chromatin binding (THOC4), an endonuclease (ENDOGL1) and an RNA-splicing factor (SYF2) were all enriched in HVC, as were genes involved in protein translation initiation and elongation (PRKRIP, RPL18A). In contrast, nucleolar proteins (
Several markers are involved in the metabolism and/or actions of steroids. For example, a corticotropin-releasing hormone binding protein (CRHBP;
Finally, we confirmed a strong HVC enrichment of zRalDH (a.k.a. ALDH1A2) and identified several large clusters of markers that have been implicated in broad aspects of retinoic acid metabolism and/or function, or that have been shown to be regulated by retinoic acid in other systems. This included markers related to gene transcription (NDRG4 and MEIS1, GTF2H1*, JUN*, and CBX3*), growth suppression and/or apoptosis (LMO2, LMO3, RHOB, NBL1, FAM5C, UBE2D3) retinoic acid metabolism (ANXA6, HSD17B11/13* - the latter consistent with the in situ pattern in
One cluster was related to angiogenesis and blood brain barrier formation. While all of the enriched genes were positive regulators of angiogenesis, genes with anti-angiogenic activity had lower expression in HVC, including ANTRX2, a positive regulator of capillary morphogenesis, and two brain-specific angiogenesis inhibitors (BAI3). A second cluster represents protein-hydrolyzing endopeptidases, and includes members of the metalloproteinase family, a membrane-bound arginine/lysine carboxypeptidase (CPM), and legumain (LGMN), a cysteine protease.
Our results provide a comprehensive classification of more than 280 genes from our primary HVC marker list that are related to a wide variety of genetic, biochemical and cellular functions as presented in
Our GO analysis revealed that the products of HVC markers are almost twice as likely to be localized to the plasma membrane as compared to a broader collection of brain-expressed cDNAs. The fact that the majority of these markers have receptor, ion channel, calcium ion binding, and G-protein signaling activities (
The discovery of bona fide markers based on the comparison of HVC vs. the underlying nidopallial Shelf provides important support for our rationale that HVC constitutes a molecular specialization of the nidopallium that is the product of specific programs of gene regulation. Furthermore, because our microarray comparison was not to the whole brain, we were able to identify markers that are also expressed in other song nuclei and brain subdivisions in various combinations (
In contrast to shared markers, the markers found to be exclusive to HVC may reflect functional properties unique to HVC. MUSTN1 (
Emulsion autoradiography revealed that some HVC markers have distinct cellular distributions, suggesting that our markers may label different cell types in HVC and provide potentially important clues about cellular specializations within the song system. Of particular interest, our results confirm that at least one HVC-enriched serotonin receptor subunit (
The large number of markers revealed by our bioinformatics analysis to be related to cellular adhesion, neurite outgrowth and extension, and cytoskeletal organization suggests that these are major targets of regulation in HVC (
Gene abbreviations are in the text and
Our results also suggest that axon guidance is an important target of regulation in HVC, and that semaphorin-related (not netrin-related) signaling is a major candidate mediator of axon guidance for HVC neurons. Follow-up
The overall balance of regulation (decreased expression in HVC) of genes involved in neurite outgrowth and extension, or the initial stages of the formation of axonal processes, suggests a decreased ability of HVC neurons to initiate neurite outgrowth and/or dendritogenesis. Consistent with this possibility, the overall balance of microtubule-associated proteins, destabilizing factors, and inhibitors of microtubule assembly suggest an increased destabilization of microtubule assembly, which is potentially linked to an increase in the stability of cell structure and a decreased ability to initiate neurite extension. Moreover, consistent with previous data
The many markers involved in the assembly, function and/or regulation of neurotransmitter receptors, included several related to peptidergic, aminergic, and cholinergic signaling, and suggested that HVC is a major target of neuromodulation by these pathways (
We also identified novel candidate peptidergic and glycinergic modulators of HVC. First, the glycinergic receptor subunit GLRA2 has very low expression in HVC, but is significantly expressed in shelf, suggesting a novel role for glycinergic transmission in parts of the adult avian pallium. This is an intriguing possibility, since the avian pallium is thought to share a common origin with the mammalian cortex, and it has been recently confirmed with electrophysiological recordings in awake zebra finches (Lovell et al, unpubl. observ.). We also found that a precursor of neurotensin/neuromedin N (NTS), as well as UTS2B, a peptide with potent vasoconstrictor activity, are both highly enriched in HVC and could represent novel peptides used by HVC neurons themselves. In contrast, several peptide receptors (
In general, our results suggest an overall decreased expression of genes in HVC that are related to cellular excitability (
In sum, the numerous markers in this category provide important clues about mechanisms that may determine the physiological and synaptic properties of HVC neurons. They also represent novel candidate targets for pharmacological manipulation that may help to address how specific cellular and synaptic properties influence the HVC and the song system. A comprehensive cellular analysis will be important to link the differential expression of specific subunits to individual cell types.
The dynamics of cell proliferation, migration, differentiation and survival play prominent roles in shaping the anatomical and functional organization of HVC throughout life. Accordingly, nearly 25% of our HVC markers were linked to these processes, including a large cluster of non-p53 related genes involved in cell proliferation and cycle progression that were enriched in HVC, and a set of genes related to tumor suppression and/or proliferation that had low HVC expression (
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.
Our study also revealed that some HVC markers are linked to cell migration or differentiation, and play important roles in the maturation and integration of post-mitotic cells into mature circuitry, and patterning during development. For example, the extracellular matrix protein TNR plays a unique role in neuronal recruitment out of the migratory stream in the olfactory bulb
Based on GO analysis, genes associated with cell nuclei, including those encoding factors with DNA-binding and transcriptional activity (
Sex steroids (i.e. androgens and estrogens) are known to play prominent roles in sexual dimorphism, the development and physiology of the song system, and adult neurogenesis. Interestingly, despite the pervasive effects of androgens in HVC, we identified only 3 genes with known androgen-related action. We found a similarly small number of genes, including CYP19A1 (i.e. aromatase), that are known to participate in the metabolism or to mediate the actions of estrogens. This might be due to the relatively small number of androgen and/or estrogen-related targets characterized to date. In light of the known distribution of androgen and estrogen receptors (predominantly in HVC and Shelf, respectively), we suggest that the identified androgen- and estrogen-related markers result from differential gene regulation in the HVC and Shelf respectively, consistent with the notion that these areas may be predominantly under androgenic and estrogenic regulation, respectively. Future analyses of genome binding sites for estrogen and androgen receptors may provide a more comprehensive method for identifying additional steroid target genes in the song system.
The Vitamin A metabolite retinoic acid has broad actions during development and cellular differentiation. In songbirds, it is required for the maturation of song and is specifically synthesized in HVC's X-projecting neurons by the enzyme zRalDH
Overall, our results provide the most comprehensive characterization to date of molecular genetic specializations of HVC that are likely related to the unique properties of this song nucleus. Specifically, our results add: (1) more than 200 novel HVC markers, including genes that may be markers of specific cell populations, (2) information about likely targets for pharmacological and/or genetic manipulations, thus providing a wealth of possible new tools that songbird researchers could use to dissect the physiology of the song system, and (3) new perspectives on the roles that individual genes and genetic pathways might play in various aspects of cellular morphogenesis, excitability, neurotransmission, neurogenesis and cell survival, steroid and retinoid metabolism and sensitivity, and gene regulation in HVC. Future studies will be directed at determining whether and/or how these genes may modulate different aspects of song production and/or learning, the main functions of song nucleus HVC.
All birds were adult male zebra finches (>120 days,
For each bird (n = 6) a set of 10 slides was selected that contained sections through HVC (∼1.4 to 2.4 mm from the midline) and the immediately adjacent nidopallial Shelf area (
HVC was identified under bright-field illumination by the characteristic bump on the brain surface and the presence of clusters of large- to medium-sized ovoid-shaped cells oriented parallel to the overlying ventricle (
We captured HVC or Shelf samples (n = 20 sections) onto CapSure Macro caps on an Arcturus PixCellII system (Mountainview, CA) mounted on a Nikon microscope at 10–20× using standard laser settings. Tissue not embedded in the cap was removed by application of CapSure strips and confirmation of captures was made by visual inspection. Caps were then placed in an Extractsure device (Arcturus), 10 µl of lysis buffer with β-mercaptoethanol (0.7 µl per 100 µl lysis buffer; Nanoprep, Stratagene) was added, and the resulting lysate retrieved. After adding 40 µl of lysis buffer, each sample was vortexed (1 min), triturated by repeated pipetting, and stored on ice. RNA was extracted using Stratagene's Absolutely RNA Nanoprep kit and assessed by spectrophotometry (ND-1000; Nanodrop). For each bird, the HVC or Shelf samples from all selected sections were pooled for further analysis.
RNA sample amplification, dye labeling, and microarray hybridization was conducted as part of Community Collaboration under the Songbird Neurogenomics Initiative and used a universal reference design
RNA samples were subjected to two rounds of linear amplification (MessageAmpII kit; Ambion). The resulting aRNAs were reverse transcribed using indirect aminoallyl incorporation and labeled with either Cy3 or Cy5. Using a universal design, dye labeling was balanced by group and each aRNA (from HVC or Shelf) was hybridized against a common reference RNA (pooled and amplified RNA from whole telencephalon of 15 male and 15 female adult zebra finches;
Each probe pair (HVC or Shelf vs. reference) was hybridized to the arrays overnight at 42°C, washed and scanned using an Axon GenePix 4000B microarray scanner, and analyzed using GenePix Pro 6.0 software to flag aberrant spots (as in
Only ∼37% of the ESTs that showed differential expression at the FDR<0.05 level had ESTIMA (songbird3) annotations that allowed for further inquiry into gene function. To confirm annotations and identify unannotated clones, we used EST alignments against the chicken genome via blat (
To facilitate the discovery of specific biological processes or molecular functions that might be targets of regulation in HVC, we used AgBase (
To identify genetic and/or biochemical pathways that might be targets of differential regulation in HVC vs. Shelf, we performed extensive database and literature searches. For some markers, groupings according to cellular/biochemical function could be easily derived from their identities (e.g., ion channels, receptors, etc). For others, Entrez Gene (
33P-labelled sense and antisense riboprobes were hybridized to serial parasagittal sections of additional (n = 4–6) adult male zebra finches followed by phosphorimager autoradiography (Typhoon 9410, GE Healthcare) for a global assessment of brain mRNA distribution, or emulsion autoradiography followed by Nissl (cresyl violet or toluidine blue) counterstaining for regional and cellular analysis. Hybridizations and washes were performed at 65°C, as previously described
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Primary FDR<0.05 HVC Markers.
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Secondary List of Differential Expressed HVC Markers.
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Previously Confirmed Markers of HVC.
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Microarray validations by in situ hybridization
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Extended search Genelist (ND).
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We thank Dr. Anda Cornea for assistance with the laser capture microdissection; the microarrays and ESTs were made available through the Songbird Neurogenomics Initiative (SoNG;