Claudio Mello, Ph.D. - US grants

DESCRIPTION (adapted from the Abstract): This is an application for a first award to study the genomic, molecular, and cellular changes that underlie the acquisition of auditory memories in the avian brain. This application is based on the observation that when songbirds are exposed to a novel conspecific song, certain immediate early genes (i.e., ZENK and c-jun) are induced in a region of the forebrain known as the caudal neostriatum (NCM). Furthermore, repeated exposure to a single song results in a long-lasting habituation of the auditory responses of NCM neurons to this song as measured electrophysiologically; the persistence of this habituation requires new protein and mRNA synthesis. The Principal Investigator seeks to examine the functional significance of the genomic response triggered by song and its possible relation to the long-term modification of the NCM cells' auditory response properties. The specific aims are to: (1) characterize further the expression of ZENK in response to song, including the identity of cells in NCM that express ZENK; (2) search for other genes induced in the NCM by song at various times when RNA synthesis has been shown to be necessary for habituation; (3) characterize and test a method to block expression of certain song-related genes in vivo; (4) determine the position of song-related genes in the genomic cascade triggered by song; and (5) examine the role of song-related genes in the long-term habituation to a particular song.

Jarvis. Lay Abstract


Vocal learning is the process by which young animals learn to imitate the vocal sounds made by their parents or a tutor. This rare trait has only been found in humans, whales, dolphins, bats and 3 groups of birds (hummingbirds, parrots and songbirds). The goal of this project is to identify and characterize the brain areas that control vocal communication in hummingbirds, as well as test whether the brains of birds that do not have vocal learning, such as chicken and pigeons, contain similar structures. The main method used is analysis of a gene that is expressed in the brain when neuronal cells fire. Utilizing a molecular technique called in situ hybridization, one can determine the exact brain cells that are involved in the perception and production of vocalizations. By comparing hearing and vocalizing animals, one can then generate high-resolution maps of brain areas that control the production of learned vocalizations.

Language acquisition, both in terms of speech production and semantics, is a fundamental aspect of the human experience and depends on vocal learning. Why is it though that so few animals have vocal learning? Do only these animals have the necessary brain structures and connections? If so, what are these structures, and how did they arise during evolution? By addressing such questions, this study may help reveal what are the brain mechanisms required for vocal learning, and potentially help understand how humans learn speech. The results should also provide a framework for studying the neurobiology of learned vocal communication in other animal groups. If the evolution of vocal learning is under strong epigenetic constraints, it is possible that humans, cetaceans and bats have also evolved similar brain structures. Alternatively, brain areas for vocal learning may have evolved as a specialization of structures present in a vocal non-learning ancestor. Results from this project are expected to throw considerable light on these questions.

[unreadable] DESCRIPTION (provided by applicant): This FIRCA proposal is for a collaborative research project between Dr. Ricardo M. Leao, of the Department of Physiology, School of Medicine of Ribeirao Preto, Sao Paulo, Brazil, and Dr. Claudio Mello of the Neurological Sciences Institute at the Oregon Health and Science University in Portland Oregon. Dr. Mello's NIH Grant (R01- DC02853; Gene regulation in auditory learning}, is the parent grant to this proposal. [unreadable] This proposal is concerned with brain circuits involved in the auditory processing of song in songbirds, more specifically the circuitry organization of the caudomedial neostriatum (NCM) in the zebra finch. The long-term goal is to elucidate the involvement of NCM in vocal communication and vocal learning. The proposed research objectives are to understand how the song processing circuits in NCM are functionally organized and whether and how these circuits are modified by experience. [unreadable] NCM is a major area within the auditory processing pathways of songbirds, comparable to portions of the auditory cortex of mammals. NCM neurons show robust electrophysiological responses to song and a long-lasting, stimulus- specific decrease ("habituation") of these responses upon repeated presentations of the same stimulus. This experience- dependent plasticity in NCM is a major candidate for a mechanism involved in song auditory memories. In addition, NCM neurons show marked song-induced expression of the transcription factor zenk. The established link between zenk and neuronal plasticity in mammals suggests that zenk expression in NCM is associated with synaptic plasticity in NCM circuitry. However, the information on the cellular and synaptic physiology of NCM required for testing hypotheses on the functions of NCM and song-induced zenk expression is currently not available. [unreadable] The proposed experiments will use electrophysiological recordings in slices in combination with morphological, tract-tracing, and immunocytochemical analysis to determine the circuit organization of NCM and to evaluate whether the habituation of NCM to song involves changes in the properties of the neuronal cells that constitute song auditory processing circuits in NCM. Results from the proposed study will contribute to our understanding of how auditory processing circuits for learned vocalizations are organized and modified by experience. Because birdsong is a learned vocal behavior, these studies will potentially help us understand how humans acquire speech, as well as possible mechanisms involved in certain speech and language disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Songbirds represent a well-established model to study vocal communication and learning and the effects of sex steroids on brain and behavior. Intriguingly, the neuronal circuitry involved in the perception, discrimination and memorization of song also expresses high levels of aromatase, an enzyme involved in the synthesis of estrogen from its precursor testosterone. This overlap between song-processing and estrogen-generating elements occurs within the caudomedial nidopallium (NCM), a central auditory area comparable to a portion of the auditory cortex of mammals. The functional consequences of the enriched expression of aromatase in NCM are unknown. The experiments in this R21 proposal are highly exploratory in nature and are designed to address two hypotheses regarding the potential role of aromatase expression in NCM: 1) We hypothesize that estrogen, both circulating, and produced locally in NCM through the action of aromatase, modulates the responsiveness of song-processing circuitry, thereby regulating the bird's response to auditory cues important for territorial and reproductive behaviors. To test this hypothesis, we will examine whether the expression levels of known and novel song-induced genes in NCM are modulated by estrogen. 2) We hypothesize that the activation of song-responsive neuronal circuitry in NCM regulates the local production of estrogen, which could have important consequences for brain physiology and behavior. To test this hypothesis, we will conduct direct measurements of aromatase activity and estrogen levels in the NCM of song-stimulated birds. Although much research in songbirds has been dedicated to the auditory system and the effects of sex steroids on brain function, potential interactions between these two systems have been largely unexplored, particularly at the level of a cortical-like area like NCM. The experiments proposed are therefore a significant departure from previous studies and will open up new avenues of investigation. Our efforts are likely to result in an improved method for directly measuring local estrogen levels in the songbird brain, as well as the potential identification of novel gene targets of estrogen regulation. The results will be highly relevant to understanding how environmental and hormonal factors interact to regulate social and reproductive behaviors in birds and other vertebrates including humans. PUBLIC HEALTH RELEVANCE Although much research in songbirds has been dedicated to the auditory system and the effects of sex steroids on brain function, potential interactions between these two systems have been largely unexplored, particularly at the level of a cortical-like area like NCM. The experiments proposed are therefore a significant departure from previous studies and will open up new avenues of investigation. The results will be highly relevant to understanding how environmental and hormonal factors interact to regulate social and reproductive behaviors in birds and other vertebrates including humans.

DESCRIPTION (provided by applicant): The study of the song control system of songbirds has provided fundamental insights into basic questions in neurobiology, including the neuronal basis of vocal learning and communication, the action of sex hormones on the brain and behavior, seasonal and adult neuronal plasticity, and the discovery of continued neuronal replacement in the vertebrate brain. Our long-term goal is to understand the functional organization of the song control system, and to determine how genes expressed in song control nuclei contribute to vocal learning, brain sex differentiation, and learning-related synaptic and neuronal plasticity. This goal has been greatly facilitated by the recent availability of glass-spotted microarrays containing ~18,000 unique brain-expressed cDNAs, a resource generated by an NINDS-funded collaborative consortium to study songbird neurogenomics (SoNG;NS045264). Using this resource, we have already identified ~200 genes that are enriched in the song nucleus HVC, a key nucleus involved in several aspects of song learning and production, and confirmed the patterns of expression for 20 of these using high-throughput in situ hybridization techniques. We now propose to extend this study and provide a comprehensive regional and cellular analysis of genes expressed in HVC and the songbird brain in general, and the pathways underlying molecular specializations within these nuclei. Our Specific Aims for this proposal are to: Aim 1: To identify molecular specializations of HVC in zebra finches. Aim 2: To determine the cellular expression patterns of molecular markers of HVC. Our proposed experiments represent the first steps towards generating a definitive molecular profiling of the oscine song control system and building a comprehensive, publicly available gene expression database for the zebra finch brain. We anticipate that this study will identify many novel molecular markers of specific song nuclei and uncover mechanisms involved in regulating the organization and function of the song control system of songbirds, and basic principles that control vocal learning, the basis for speech and language acquisition in humans. PUBLIC HEALTH RELEVANCE The identification of molecular markers and pathways underlying cellular specializations of specific song nuclei will likely uncover mechanisms that regulate the organization and function of the song control system of songbirds, and basic principles that control vocal learning, the basis for speech and language acquisition in humans. Our research may therefore help elucidate basic mechanisms of speech and language disorders. In a broader sense, however, our results are also likely to identify basic principles underlying brain sexual differentiation, learning and memory, and the control of brain plasticity and repair, including neuronal replacement during adulthood.

DESCRIPTION (provided by applicant): Songbirds are a leading model of neurobiological research with wide implications for understanding issues of human health and disease. They are among the few organisms that have evolved vocal learning, a complex trait that provides the basis of spoken language acquisition in humans. Studies of the ontogeny of songbird vocalizations and the organization of the brain circuitry that controls song learning and production have provided unique opportunities for uncovering the neural bases of vocal learning. Songbird research has also contributed novel insights into a broad range of fundamental questions in neurobiology, such as behaviorally- regulated gene expression, sex dimorphisms and the effects of sex steroids on brain structure and function, photoperiodicity and the regulation of seasonal brain plasticity, the role of sleep in learning, the neuroendocrine regulation of reproductive and social behaviors, and neurogenesis and neuronal replacement in adulthood. To help understand how the song control circuitry and birdsong behavior are shaped by genetic mechanisms, a wide set of modern molecular and genomic resources have recently become available to songbird researchers through NIH-funded initiatives; such resources include normalized brain cDNA libraries, comprehensive annotated EST databases, microarrays, a BAC library and the completed the zebra finch genome. Such resources have been instrumental in the rapid identification of genes and gene families of neurobiological interest, the study of gene structure and regulatory domains, high- throughput analysis of gene regulation through molecular profiling studies, and comparative genomics across the major higher vertebrate groups. A key next step in making full use of these genomic resources and understanding how genes relate to brain function and behavior in songbirds is to map gene expression in the context of functional brain circuits. To achieve this goal, we propose a single Specific Aim, namely to generate a Gene Expression Brain Atlas of the Zebra Finch. Specifically we propose to map the brain expression of a large set of genes (~2,500) that are of key importance to songbird and avian brain researchers, in register with a histological atlas, and make the data available as a web-based resource.

Hummingbirds, songbirds and parrots learn their vocalizations from adults, just like human infants learn to speak by imitating their parents' speech. This capacity provides the basis for how humans acquire speech and language, yet how the brain achieves this goal is unknown. Using an array of powerful techniques, this collaborative project will examine the anatomical, electrical, and molecular properties of brain circuits that control vocalizations in hummingbirds and songbirds, comparing them with each other and with prior human studies. Understanding how these different organisms evolved brain circuits to accomplish similar goals will reveal insights into fundamental properties of vocal learning systems. Traditional lab animals cannot be used as they lack vocal learning; hence, the use of vocal learner birds is critical. The project will provide training in multiple research techniques to high school, undergraduate, and graduate students, emphasizing underrepresented groups. It will also promote broad dissemination of findings and outreach activities, related to both scientific and conservation efforts. The project is also integrated with an International Consortium (funded by the Brazilian Government) for cataloguing and characterizing the diversity of tropical birds, including integration of Museum collections, generation of genome sequences, and examination of brain specimens relevant to the evolution of vocal learning. These activities will enable interactions between US and Brazilian faculty and students, while promoting training in molecular and histological methods through site visits, field trips, and workshops.

The vocal control system of songbirds is critical for song production and learning, and is well characterized anatomically, electrophysiologically, and molecularly. However, knowledge of the analogous areas in other avian vocal learners is limited. Recent phylogenomics efforts reveal that hummingbirds evolved vocal learning independently of songbirds; thus, comparing their vocal control systems will reveal convergently evolved features that may be fundamentally required for this trait. The investigators will use tract-tracing to determine how vocal control areas are connected in hummingbirds, in vitro electrophysiological recordings to determine intrinsic neuronal properties of vocal areas in hummingbirds and songbirds, and in situ hybridization to identify molecular markers of vocal nuclei. Evidence of shared anatomical, physiological and molecular specializations will point to convergent features representing possible universal properties of vocal learning systems that may also be shared with humans. Alternatively, differences would suggest that multiple circuit and cellular/molecular architectures can subserve vocal learning. Outcomes will provide novel clues as to evolutionary origins and constraints of vocal learning and associated pathways, leading to insights into fundamental requirements of vocal learning.

? DESCRIPTION (provided by applicant): The availability of NIH/NHGRI-funded high quality genomes for non-human vertebrates, including avian species like the zebra finch and chicken, has made it possible to search for genomic features that are associated with traits whose mechanisms cannot be investigated in humans, thus requiring experimental model organisms. The study of some complex learned behaviors and their associated brain circuits illustrate well this point. We propose here a set of exploratory analyses in the zebra finch, a songbird, in order to elucidate the genomic basis of vocal learning, a complex behavioral trait that is a prerequisite for speech and language acquisition in humans. Zebra finches have emerged as the premier model organism for investigating the biological basis of vocal learning. In fact, no other organism, including rodents and non-human primates, provides a comparable experimental platform that allows investigators to relate neural and genomic features to a learned vocal behavior with close ties to human speech. Recent studies have revealed remarkable convergent molecular specializations in brain circuits for vocal control in songbirds and humans, pointing to a set of shared molecular requirements for birdsong and human speech learning. Such findings have broad implications for understanding genetic mechanisms that may underlie a variety of human communication impairments and disorders. Here we propose to implement a novel computational search algorithm that is designed to discover novel and duplicated genes in the genome of the zebra finch that are absent in a closely related vocal non-learning species (manakin), and thus possibly related to vocal learning. We anticipate that this exploratory effort will identify ~100 novel genes in zebra finches. To further test for a possible link to vocal learning, we will examine the occurrance of the identified genes in multiple other vocal learner birds whose genomes are now also available. An exploratory expression analysis will attempt to link the novel genes to specific brain cell types associated with vocal learning, as well as with other songbird traits of relevance to human health, including adult neurogenesis, brain dimorphisms and sex steroid action, and sleep modulation of learning. We will also explore whether expansions of functional protein domains in the novel genes are present in other avian vocal learner species, as well as in humans. If found in the latter, such features could potentiall be used in future studies of genetic correlates of speech proficiency and disorders. The outcomes will also guide future mechanistic studies to establish causal links between specific genes and vocal learning. Lastly, our intent is to implement a computational search algorithm of broad applicability that can be utilized for novel gene searches and genome curations in any target species of interest; such an algorithm should therefore be of broad use, particularly for analysis of recently assembled genomes that have low quality or no annotations.

Project Summary Avian model organisms, including songbirds (zebra finch, canaries, starlings), chicken, quail, and pigeon have contributed much to our understanding of brain function and of disorders that affect neural development, function, and cognition. However, we still lack a clear understanding of how avian brain structures, particularly those that subserve complex learned behaviors and cognition, relate to brain structures in mammals, including humans. It is also unclear how molecular brain specializations of birds relate to those of mammals. To address these gaps, we used funding from the NINDS (R03) and NIGMS (R24) to develop the Zebra finch Brain Expression Atlas (ZEBrA), currently the only in situ hybridization database of brain gene expression for any avian species. ZEBrA is a publicly accessible website that contains >2,200 high resolution digital images of brain sections from adult male zebra finches that are aligned to a reference histological atlas, and hybridized to reveal the expression of >500 genes of relevance for brain development, physiology, plasticity, and learning, including numerous linked to human diseases or to lethal or deleterious phenotypes in rodents. Many of the patterns in ZEBrA reveal differential expression across broad brain subdivisions, previously unsuspected subdomains that cannot be visualized with conventional histology, and high enrichment in nuclei within circuits underlying specific behaviors (e.g., the system that controls vocal production and learning). These patterns have yielded novel insights into molecular specializations of major regions and of specific nuclei, including the discovery of convergent molecular specializations of vocal areas that are shared between songbirds and humans. The availability of ZEBrA has had a large impact (>4,000 users, and 35,000 page views), providing an important source of genetic and neuroanatomical data for a large number of songbird and avian researchers, many funded by the NIDCD, NIMH, NINDS or NICHD. The present proposal aims to use NIGMS's Legacy mechanism to maintain ZEBrA during a transition phase where further funding is sought to ensure the long- term availability, maintenance, and possible future expansion of this unique resource. Such studies will help to further validate the use of avian model organisms for understanding the molecular basis of brain function and disorders. Planned activities consist of regular updates to ZEBrA to ensure links to important databases and compatibility to browsers are kept up-to-date, and addition of 300 already processed genes whose images are not yet available online. Included are also plans for broad dissemination of the resource, close interactions with the community to ensure its needs are met, and evaluation to maximize ZEBrA's utility and impact. Legacy funding will facilitate the transition effort, which includes the preparation of applications to an array of NIH institutes whose stated missions are in line with ZEBrA's goals, and a more limited effort to generate brain expression data for specific genes of interest to individual users but not yet on the database, on a pay for service basis.

Over the past four decades, advances in gene manipulation technologies have dramatically improved our understanding of numerous fields in biology. However, although studies in birds have made seminal contributions to fields such as development, neurobiology, and immunology, bird research has been hindered by the limited availability of gene manipulation tools, including the ability to make transgenic birds. In other model species, such as mice, zebrafish, and fruit flies, it is much easier to generate transgenic animals that directly assess the role of a particular gene of interest. Some of the most successful transgenic technologies rely on gene-editing and isolating and modifying stem cells, and then transplanting them into hosts. In contrast, in birds, the advanced age of embryos at the time eggs are laid, and the lack of efficient viral vector tools has greatly limited gene manipulation efforts. To address these limitations, the investigators develop and improve gene manipulation and stem cell technologies in birds. The project focuses on the zebra finch, a vocal learning songbird that is the most commonly used animal model to study the neural and genetic basis of human speech and language. Since other animal models commonly used in research do not have vocal learning, this is the first time that efficient methods for gene manipulation are being developed for a vocal learner species. Other beneficiaries include all avian research and possibly any egg-laying species, many of which are used to address questions in diverse areas of biology. The project also provides research opportunities for high school, undergraduate, and graduate students and outreach to communities typically underrepresented in science and technology fields.

To provide gene manipulation tools and protocols that will be useful not only to the songbird research community, but also to avian researchers in general, the investigators are: 1) generating transgenic zebra finches that express the genome-editing enzyme Cas9 under a ubiquitous promoter; 2) isolating, culturing, and utilizing primordial germ cells (PGCs) to improve the efficiency of generating transgenic zebra finches; and 3) developing efficient viral vectors for manipulating genes in zebra finch cells. Using established methods transgenic zebra finches are made by injecting VSV-pseudotyped lentiviral vectors into freshly-laid fertilized eggs. When combined with CRISPR-designed guide RNAs, these transgenic songbirds enable gene-editing capabilities in a variety of tissues and cell types. To scale up and create many transgenic lines, PGC culture methods are being optimized and viral vectors with higher transfection rates in zebra finch cells developed, thus reducing the materials, time, and animals needed to create a new transgenic line, as well as greatly facilitating gene manipulations. The tools and optimized methods generated by this project will also impact avian research in fields other than birdsong biology. PGC culturing protocols can be applied to other avian species, viral vectors optimized for specific songbird tissues may also have higher transfection rates in analogous cell types of other avian species, and the Cas9-lentiviral construct could be used to generate other strains of Cas9-expressing transgenic birds.

Non-Technical Paragraph:
This proposal funds travel for a group of NSF-invited genomics researchers to attend the "Perspectives in Comparative Genomics & Evolution" meeting being jointly organized by NIH-NHGRI/USDA/NSF that will take place Aug. 15-16, 2019 at the Marriott North Bethesda. This meeting will address growing opportunities as well as challenges in the fields of Evolutionary and Comparative Genomics. It includes presentations and discussions about the genomes of humans, traditional and non-traditional lab organisms, and agricultural and wild species, as well as how their comparative study can help scientists to devise strategies that can better impact human health, agriculture and conservation practices. Invited speakers are recognized authorities and emerging scientists who are applying cutting-edge genomics and bioinformatics tools to address a broad range of fundamental questions in evolutionary genomics. This interagency conference is intended to identify effective strategies that can broaden the impact of comparative genomics research by targeting research efforts that impact basic, applied, and health focused systems. Speakers and attendees are being invited paying close attention to balanced representation of gender and seniority level, as well as the need to cover the broadest possible theme range. Among the expected outcomes of this conference will be the production of one or more white papers, as well as an interagency position statement. These documents will be shared with the general scientific community, outside stakeholders, as well as program officers in each of the participating federal agencies.

Technical Paragraph:
The explosive growth of available genomic data and related resources for a broad range of species, including humans, model organisms, species of agricultural relevance, and wildlife species, has created opportunities to better understand basic principles of comparative genomics and evolution, as well as potential applications to health, agriculture, and conservation. Challenges include the need to develop methods that can handle vast amounts of data, assurances for high quality of genomes and feature annotations, and the development of effective computational and bioinformatics approaches that take into account phylogenetic principles. This meeting will address these needs by bringing together experts with diverse backgrounds, expertise, and perspectives in comparative genomics. The format includes series of short talks on key themes, as well as extensive follow-up discussions and targeted breakout sessions, maximizing the open exchange of novel approaches and emerging solutions to shared problems. Expected outcomes include identifying areas of synergy, current gaps in knowledge and resources, as well as defining areas of relevance to the NIH/NHGRI for understanding human health and disease. Partnerships with the USDA and NSF will place further emphasis on the relevance of species that are of agricultural and economic relevance, as well as the pressing need for genomics research on wildlife species. Overall, the planned in-depth discussions will help to identify aspects of comparative genomics that should be at the "forefront" of this burgeoning field, meeting the demands of a range of stakeholders.

This award was co-funded by the Enabling Discovery throughout GEnomic (EDGE) Program and the Plant Genome Research Program (PGRP) in the Division of Integrative Organismal Systems and the Infrastructure Innovation for Biological Research (IIBR) Program in the Division of Biological Infrastructure.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Project Summary Zebra finches (Taeniopygia guttata) are an important non-traditional avian model organism that have made major contributions to a range of questions with important implications for human health and disease. Funded by several NIH Institutes (e.g. NINDS, NIDCD, NIMH, NIGMS, NHGRI, and NIDA), studies in finches have addressed such topics as vocal communication, endocrine modulation of reproductive and social behaviors, sex steroids actions in brain and behavior, sex dimorphisms, the role of sleep in learning, motor coding and rhythms, mechanisms of hearing function, the dynamics of adult neurogenesis and neuronal replacement, and the role of endocannabinoids in cell signaling and addiction, among other themes. Finches are also the prime model organism for studying the neuronal and genetic basis of vocal learning, a trait that provides the basis of human speech and language learning and that is absent or rudimentary in rodents and monkeys. A major bottleneck to finch studies, however, has been the lack of high efficiency tools for gene manipulation, which has limited the ability to establish causal links between genes and phenotypes of interest in these birds. VSV- G-pseudotyped lentiviruses (VSV-G-LVs) are routinely used in gene manipulation studies because they readily transduce a broad range of tissues and organisms, however they show very low transduction in finches. We have recently found that the receptor for VSV-G-LVs, namely the low density lipoprotein receptor (LDLR), is a pseudogene in finches, providing a likely explanation for the low LV transduction. We have also obtained evidence that finches have evolved unique features of cholesterol metabolism that may compensate for this gene loss, consistent with a central and conserved role for LDLR in cholesterol uptake, as well as deleterious effects on health when this gene is disrupted in humans (e.g. increased risk for coronary disease, genetic hypercholesterolemia). Importantly, we have found that expressing human LDLR (hLDLR) in cultured finch cells markedly boosts VSV-G-LVs transduction, pointing to a plausible solution for the low VSV-G-LV efficacy. In this exploratory R21, we propose to: SA1) Determine the effectiveness of LDLR constructs for VSV-G- LV transduction. We will use an in vitro assay with cultured finch cells to examine how hLDLR-based constructs that vary in the domains for viral binding or receptor recycling affect VSV-G-LV transduction; and SA2) Generate and characterize zebra finch transgenic lines harboring a functional hLDLR. We will use established protocols to generate and characterize transgenic finches that constitutively and ubiquitously express LDLR constructs engineered to maximize VSV-G-LV transduction. We predict that these transgenic lines will show high VSV-G-LV transduction across multiple tissues, making them important resources for future gene manipulation and CRISPR-based gene editing applications that address a wide range of biomedical questions. They will also serve as novel tools for better understanding the genetics and evolution of LV infectivity and cholesterol metabolism in vertebrates.

Project Summary Avian model organisms, including songbirds (zebra finches, canaries, starlings), parrots, chicken, quail, and pigeons have contributed much to our understanding of brain function and disorders that affect neural development, function, and cognition. Furthermore, many bird groups are being increasingly recognized as having enlarged brains that are capable of advanced cognitive and learning skills that rival and even surpass those in mammals. Despite these contributions, we still lack a clear understanding of how the molecular brain organization in birds compares to that in mammals, including humans. To address this gap, we utilized resource building funds from the NINDS and NIGMS to develop the Zebra finch Expression Brain Atlas (ZEBrA), currently the largest in situ hybridization database of brain gene expression for any avian species. ZEBrA is a publicly accessible website with a database containing >3,500 high resolution digital images of brain sections from adult male zebra finches that are aligned to a reference histological atlas, and hybridized to reveal the brain-wide expression of >720 genes of relevance for brain development, physiology, plasticity, and vocal learning. Notably, nearly 200 of these genes have been linked to speech and/or neural disorders in humans, and/or constitute shared molecular specializations of analogous brain regions for vocal production and learning in birds and humans. Many expression patterns in ZEBrA have also revealed previously unsuspected subdomains that are not visible with conventional histological techniques, as well as enrichments in discrete nuclei within circuits that underlie specific behaviors (e.g., vocal production and learning). Despite these findings, a quantitative analysis of the ZEBrA database has not yet been performed, which has hindered our ability to perform accurate comparative analyses with similar resources from mammals (e.g. Allen Institute's Mouse Brain Atlas - MBA). We propose here to use image analysis methods to extract equivalent regional gene expression data from both databases. The outcomes in finch will define regional molecular profiles of major brain areas and specialized nuclei of the vocal control and learning circuitry, the latter a cognitive trait of high relevance to human speech and language. The ZEBrA and MBA data will also be compared to derive insights into how avian and mammalian brains relate or diverge molecularly. Such insights will help to further validate the use of avian species as informative model organisms for understanding the molecular basis of brain function and cognitive skills, as well as the genetic basis of brain disorders of high relevance to humans.