For decades, songbirds have served as a premier model for conducting neurobiological research with wide implications for understanding human health and disease. Through a series of NINDS-, NHGRI- and NIGMS-funded projects, a large collection of molecular and genomic tools and resources have been made available in zebra finches and have begun to shed light on the basic molecular and genetic mechanisms that regulate the song control system and singing behavior. These resources include normalized brain cDNA libraries, comprehensive annotated brain EST databases, a BAC library, and the sequencing, assembly and annotation of the zebra finch genome. To make full use of these resources and for understanding how genes relate to brain function and behavior in songbirds, it is critical to map brain gene expression in the context of functional circuits. The goal of the ZEBrA project is to develop a publically available on-line digital atlas that documents the expression of a large collection of genes within the brain of adult male zebra finches.
ZEBrA is being constructed in the Mello laboratory (Dept. of Behavioral Neuroscience, OHSU, Portland OR). All of the genes in ZEBrA are named according to their orthology with unique human loci and assigned a name (or gene symbol) that has been established and is curated by the Human Genome Nomenclature Consortium (HGNC). All of the clones used for this project are derived from normalized zebra finch brain cDNA libraries and are part of the Songbird:ESTIMA collection (clones available upon request). DIG-labeled antisense riboprobes are hybridized to parasagittal brain sections derived from adult male zebra finches using an optimized non-radioactive in situ hybridization protocol specifically developed for ZEBrA. Genes with highly differential expression patterns are hybridized to several sections (5-9 levels, ~0.2 - 4.0 mm from midline), while genes with non-differential patterns are hybridized to 1-4 levels (~1.0 and 2.4 mm from midline) focusing on documenting expression in song nuclei; all brains are from confirmed awake, non-singing and non-stimulated birds. Sections are imaged with a digital scanner (Olympus Nanozoomer) by the Mitra lab at Cold Spring Harbor Laboratory, or a Aperio AT2 scanner (Leica) by the OHSU Knight BioLibrary. Images are presented in reference to a set of corresponding drawings derived from a histological atlas of the zebra finch brain, as described in Karten et al. (J. Comp. Neurol. 2013; 521(16):3702-15). These drawings are not intended as matching schematics for the in situ data, but do provide an approximation of the brain level and corresponding structures with labels for each individual in situ images. The nomenclature is based on the revised nomenclature as described by Reiner et al. 2004, with further modifications as proposed in Chen et al. 2013 and Jarvis et al. 2013.
ZEBrA includes various sets of genes that are of relevance to birdsong neurobiologists, but also to those interested in human speech and language mechanisms and disorders, the use of animal models for understanding human genetic diseases with neurological and psychiatric components, as well as comparative neuroanatomy and brain evolution. Many of the genes in ZEBrA were initially identified through molecular screenings of specific song nuclei, but other genes represent our efforts to cover important gaps in our understanding of the molecular and neurochemical properties of the song system, clarify molecular correspondences between avian and mammalian brains, and address specific needs from the songbird and avian brain research communities. The main categories of ZEBrA genes correspond to the major ZEBrA portals, as detailed below:
(1) Markers of the Song System. Many of the genes presented in this portal were initially identified through microarray screening and deep sequencing efforts focused on specific song nuclei. Other genes, including many that can also be found in other ZEBrA portals, were discovered to be song system markers by close inspection of their expression patterns in the song system. Regardless of how they were discovered, the genes presented in this portal are differentially expressed in song nuclei compared to adjacent brain areas, and represent molecular specializations that may define unique properties of the song system. These genes are also neuroanatomical markers that help to define nuclear boundaries, allowing greater precision in studies of nuclear size, shape, or organization that occur naturally or as a result of experimental manipulations.
(2) Speech and Language. Like humans, zebra finches are vocal learners and have a specialized circuitry that subserves this behavior. Zebra finches are a major model organism for studying the biological basis of vocal learning, one of the foundations of human speech and language capabilities. The genes presented in this portal all have an established or hypothesized involvement in human speech and language production mechanisms and disorders.
(3) Diseases and Phenotypes. A significant number of genes in ZEBrA are associated with genetic disorders or phenotypes that affect brain function and/or behavior. One set of genes presented in this portal was selected based on established links between their human orthologs and specific genetic disorders in humans, particularly those with major neurological or psychiatric components, as described by the Online Mendelian Inheritance in Man (OMIM) database. A second set of genes corresponds to genes whose mouse orthologs are associated with neurological or behavioral phenotypes, as described by the Mouse Genome Informatics (MGI) database. The ZEBrA expression patterns of genes in this portal allow for an exploration of disease-related genes and pathways in the context of avian brain structures and circuits.
(4) Comparative Neuroanatomy. Comparative analyses offer unique opportunities for understanding how brains with distinct organizations can subserve similar aspects of brain function and behavior. One set of genes presented in this portal consists of markers of specific structures and/or subdivisions of the zebra finch brain. A second set of genes consists of markers that are selectively or differentially expressed in different parts of the mammalian brain (e.g., different cortical layers, amygdalar nuclei, hippocampal subdivisions, etc). Close comparative examination of these expression patterns can help to shed light on similarities and differences in the organization of mammalian and avian brains. Specifically, this comparison will help determine the extent to which molecular specializations of homologous brain structures are shared between mammals and birds, and yield further insights into the relatedness of regions whose homologies are contentious.
(5) Gene Function. ZEBrA genes in this portal are organized into families and groups that are known from studies in other organisms to be associated with various key aspects of brain function and neuronal physiology. These include genes underlying properties of cell excitability (e.g., neurotransmitter/neuromodulator receptors, ion channels, regulators of synaptic function), axonal guidance, gene regulation (transcription factors), metabolic pathways, and intracellular signaling cascades, among others.
(6) Songbird Discoveries. Since its release, ZEBrA has aided researchers in numerous studies and contributed several novel fundamental insights. In this portal we showcase select papers that have taken advantage of ZEBrA's extensive brain gene expression database and unique brain atlas features. We also present the zebra finch brain expression patterns of genes that are highlighted in published songbird studies. Examination of these patterns can help in the discovery of links between genes, brain organization, seasonality, sex dimorphism, steroid regulation, neuronal plasticity, vocal learning, neurogenesis, and many other basic neurobiological themes.
Funding for Project ZEBrA
Project ZEBrA was initially funded by a pilot grant from the NIH/NINDS (R03-NS059755), and subsequently directly funded by resource grants from the NIH/NIGMS (R24 GM120464-02 and R24-GM120464) to CVM. Project ZEBrA also benefits from a grant from the NIH/NINDS in support of the Song Neurogenomics Consortion (SoNG; R01-NS045264), which generated the brain EST/cDNA clone collection from which all in situ probes for ZEBrA are derived. ZEBrA has also benefitted from funding to the Mello lab from NIH/NIDCD (R21-DC014432) and from NSF (IOS-143602) to conduct genomics, transcriptomics, and brain gene expression analyses in zebra finches and other avian vocal learning species (Anna's hummingbirds and budgerigars).
Current Team ZEBrA (Mello lab)
Claudio V. Mello - Principal Investigator
Peter V. Lovell - Lead ZEBrA Developer and Project Manager
Taylor Kaser - in situ database
Alexa Buckner - Image processing, Database management
ZEBrA Marker Annotators
Imaging Collaborators (Mitra lab)
Partha Mitra - Imaging consultant and resources
Vadim Pinskiy - Imaging Coordinator
Alex Tolpygo - Imaging Coordinator
Former Team ZEBrA members
Brian R. Snider - Programming
Julia Carleton – Genome annotations, In situ database
Tessa Marzulla – Genome annotations, In situ database
Anne McHugh – In situ database
Katy Horback – In situ database
Michelle Hribar – Programming
Daniel Tang - Histological Atlas preparation
David F. Clayton - UIUC
Anton J. Reiner - UTHSC
Harvey J. Karten – UCSD
Partha Mitra - CSHL
Michelle Hribar - OHSU