Constance Scharff

The genome of a songbird.

The zebra finch is an important model organism in several fields with unique relevance to human neuroscience. Like other songbirds, the zebra finch communicates through learned vocalizations, an ability otherwise documented only in humans and a few other animals and lacking in the chicken—the only bird with a sequenced genome until now. Here we present a structural, functional and comparative analysis of the genome sequence of the zebra finch (Taeniopygia guttata), which is a songbird belonging to the large avian order Passeriformes. We find that the overall structures of the genomes are similar in zebra finch and chicken, but they differ in many intrachromosomal rearrangements, lineage-specific gene family expansions, the number of long-terminal-repeat-based retrotransposons, and mechanisms of sex chromosome dosage compensation. We show that song behaviour engages gene regulatory networks in the zebra finch brain, altering the expression of long non-coding RNAs, microRNAs, transcription factors and their targets. We also show evidence for rapid molecular evolution in the songbird lineage of genes that are regulated during song experience. These results indicate an active involvement of the genome in neural processes underlying vocal communication and identify potential genetic substrates for the evolution and regulation of this behaviour.

FOXP2 as a molecular window into speech and language.

Rare mutations of the FOXP2 transcription factor gene cause a monogenic syndrome characterized by impaired speech development and linguistic deficits. Recent genomic investigations indicate that its downstream neural targets make broader impacts on common language impairments, bridging clinically distinct disorders. Moreover, the striking conservation of both FoxP2 sequence and neural expression in different vertebrates facilitates the use of animal models to study ancestral pathways that have been recruited towards human speech and language. Intriguingly, reduced FoxP2 dosage yields abnormal synaptic plasticity and impaired motor-skill learning in mice, and disrupts vocal learning in songbirds. Converging data indicate that Foxp2 is important for modulating the plasticity of relevant neural circuits. This body of research represents the first functional genetic forays into neural mechanisms contributing to human spoken language.

FoxP2 Expression in Avian Vocal Learners and Non-Learners

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.

Targeted Neuronal Death Affects Neuronal Replacement and Vocal Behavior in Adult Songbirds

In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.

A molecular neuroethological approach for identifying and characterizing a cascade of behaviorally regulated genes

Songbirds have one of the most accessible neural systems for the study of brain mechanisms of behavior. However, neuroethological studies in songbirds have been limited by the lack of high-throughput molecular resources and gene-manipulation tools. To overcome these limitations, we constructed 21 regular, normalized, and subtracted full-length cDNA libraries from brains of zebra finches in 57 developmental and behavioral conditions in an attempt to clone as much of the brain transcriptome as possible. From these libraries, ≈14,000 transcripts were isolated, representing an estimated 4,738 genes. With the cDNAs, we created a hierarchically organized transcriptome database and a large-scale songbird brain cDNA microarray. We used the arrays to reveal a set of 33 genes that are regulated in forebrain vocal nuclei by singing behavior. These genes clustered into four anatomical and six temporal expression patterns. Their functions spanned a large range of cellular and molecular categories, from signal transduction, trafficking, and structural, to synaptically released molecules. With the full-length cDNAs and a lentiviral vector system, we were able to overexpress, in vocal nuclei, proteins of representative singing-regulated genes in the absence of singing. This publicly accessible resource http://songbirdtranscriptome.net can now be used to study molecular neuroethological mechanisms of behavior.

Challenges for brain repair: insights from adult neurogenesis in birds and mammals.

Adult neurogenesis is a widespread phenomenon occurring in many species, including humans. The functional and therapeutic implications of this form of brain plasticity are now beginning to be realized. Comparative approaches to adult neurogenesis will yield important clues about brain repair. Here, we compare adult neurogenesis in birds and mammals. We review recent studies on the glial identity of stem cells that generate new neurons, the different modes of migration used by the newly generated neurons to reach their destinations, and how these systems respond to experimentally induced cell death. We integrate these findings to address how comparative analysis at the molecular level might be used for brain repair.

Mutations in the polyglutamine binding protein 1 gene cause X-linked mental retardation.

We found mutations in the gene PQBP1 in 5 of 29 families with nonsyndromic (MRX) and syndromic (MRXS) forms of X-linked mental retardation (XLMR). Clinical features in affected males include mental retardation, microcephaly, short stature, spastic paraplegia and midline defects. PQBP1 has previously been implicated in the pathogenesis of polyglutamine expansion diseases. Our findings link this gene to XLMR and shed more light on the pathogenesis of this common disorder.

Gene Expression Changes in the Course of Neural Progenitor Cell Differentiation

The molecular changes underlying neural progenitor differentiation are essentially unknown. We applied cDNA microarrays with 13,627 clones to measure dynamic gene expression changes during the in vitro differentiation of neural progenitor cells that were isolated from the subventricular zone of postnatal day 7 mice and grown in vitro as neurospheres. In two experimental series in which we withdrew epidermal growth factor and added the neurotrophins Neurotrophin-4 or BDNF, four time points were investigated: undifferentiated cells grown as neurospheres, and cells 24, 48, and 96 hr after differentiation. Expression changes of selected genes were confirmed by semiquantitative RT-PCR. Ten different groups of gene expression dynamics obtained by cluster analysis are described. To correlate selected gene expression changes to the localization of respective proteins, we performed immunostainings of cultured neurospheres and of brain sections from adult mice. Our results provide new insights into the genetic program of neural progenitor differentiation and give strong hints to as yet unknown cellular communications within the adult subventricular zone stem cell niche.

The songbird syrinx morphome: a three-dimensional, high-resolution, interactive morphological map of the zebra finch vocal organ

BackgroundLike human infants, songbirds learn their species-specific vocalizations through imitation learning. The birdsong system has emerged as a widely used experimental animal model for understanding the underlying neural mechanisms responsible for vocal production learning. However, how neural impulses are translated into the precise motor behavior of the complex vocal organ (syrinx) to create song is poorly understood. First and foremost, we lack a detailed understanding of syringeal morphology.ResultsTo fill this gap we combined non-invasive (high-field magnetic resonance imaging and micro-computed tomography) and invasive techniques (histology and micro-dissection) to construct the annotated high-resolution three-dimensional dataset, or morphome, of the zebra finch (Taeniopygia guttata) syrinx. We identified and annotated syringeal cartilage, bone and musculature in situ in unprecedented detail. We provide interactive three-dimensional models that greatly improve the communication of complex morphological data and our understanding of syringeal function in general.ConclusionsOur results show that the syringeal skeleton is optimized for low weight driven by physiological constraints on song production. The present refinement of muscle organization and identity elucidates how apposed muscles actuate different syringeal elements. Our dataset allows for more precise predictions about muscle co-activation and synergies and has important implications for muscle activity and stimulation experiments. We also demonstrate how the syrinx can be stabilized during song to reduce mechanical noise and, as such, enhance repetitive execution of stereotypic motor patterns. In addition, we identify a cartilaginous structure suited to play a crucial role in the uncoupling of sound frequency and amplitude control, which permits a novel explanation of the evolutionary success of songbirds.

Site-Specific Retinoic Acid Production in the Brain of Adult Songbirds

The song system of songbirds, a set of brain nuclei necessary for song learning and production, has distinctive morphological and functional properties. Utilizing differential display, we searched for molecular components involved in song system regulation. We identified a cDNA (zRalDH) that encodes a class 1 aldehyde dehydrogenase. zRalDH was highly expressed in various song nuclei and synthesized retinoic acid efficiently. Brain areas expressing zRalDH generated retinoic acid. Within song nucleus HVC, only projection neurons not undergoing adult neurogenesis expressed zRalDH. Blocking zRalDH activity in the HVC of juveniles interfered with normal song development. Our results provide conclusive evidence for localized retinoic acid synthesis in an adult vertebrate brain and indicate that the retinoic acid-generating system plays a significant role in the maturation of a learned behavior.