Kristin K. Baldwin - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The aim of this project is to reveal the neuroanatomical organization of circuits that underlie olfactory processing. While studies across species have revealed highly conserved olfactory sensory maps in the periphery, far less is known about the circuits that enable olfactory coding and perception in the brain. The primary objective of this project is to map the connectivity of input specific olfactory circuits from the nose to the cortex in the mouse brain. Findings from these studies can consequently be applied to identify cortical sites of integration for distinct olfactory inputs and to dissect the relative contribution of genes and experience in olfactory neural circuit formation and function. These studies should have implications for understanding innate behaviors such as fear and mating and also facilitate the study of learning and memory.

DESCRIPTION (provided by applicant): We aim to develop an innovative approach to generate, at high-throughput, isogenic induced pluripotent stem cells (iPSCs), and use their differentiated progeny to understand the impact of human genetic variation on the risk of developing coronary artery disease (CAD). A genomic variant associated with CAD, myocardial infarction (Ml), abdominal aortic aneurysm, and intracranial aneurysm is found in a stretch of chr9p21 devoid of known genes. We will use this locus as a model for our study; while focusing on 9p21, our overall approach will be broadly applicable to the study of HLBS diseases of complex genetic architecture.

DESCRIPTION (provided by applicant): Human cognition and behavior depend on the proper assembly and maintenance of neural circuits and genes that imperil these processes are increasingly linked to autism, schizophrenia and other neurological disorders. Neural activity and genetic programs contribute to different aspects of neural circuit development and architecture. In some sensory circuits, such as vision, hearing and touch, neurons with related response properties are organized in a stereotypic manner to form maps of sensory information. In these systems, genes and neural activity play complementary roles in forming and maintaining these maps. Less is known about the developmental principles that help to establish finer scale neural connectivity, particularly in processing regions that lack obvious spatial maps. Here, we propose to examine the impact of neural activity on circuits involved in the sense of smell. In particular, we will study the circuits formed by the mitral and tufted (MT) neurons, which receive inputs from defined groups of sensory neurons and transmit this information to multiple functionally distinct cortical processing centers responsible for olfactory learning and innate behaviors such as attraction or fear. MT neurons are important to study for two reasons. First, we have developed new viral tracing and neuron reconstruction techniques to label sets of sister MT neurons that respond to the same odors in the same animal and map their projections in three dimensions. This affords us a sensitive method to investigate fine scale wiring mechanisms. Second, individual MT neurons innervate multiple cortical targets, which each seems to posses a characteristic architecture. This allows us to simultaneously assess the impact of activity on multiple circuit architectures that are formed by different branches of the same MT axon. The central hypothesis of this proposal is that activity in MT neurons differentially regulates distinct aspects of olfactory circuit architecture. To test this hypothesi we propose three specific aims. First, we will determine whether projections of sister mitral and tufted (MT) neurons are stereotyped in cortical processing centers involved in innate behaviors. Second, we will synaptically silence olfactory sensory neurons and identify the features of olfactory bulb and cortical circuits that depend on sensory input. Third, we will synaptically silence MT neurons and identify aspects of local and cortical circuit architecture that depend on MT activity. Results of these studies will identify unknown activity-dependent mechanisms of olfactory neural circuit formation and predict which features of circuit architecture are likely tobe controlled by genetic programs. Defining these circuits with high resolution will facilitate future studies to identify genes implicated in building specific neural circuits and to assess the functional consequence of mutations linked to human neurodevelopmental or neurodegenerative diseases.

DESCRIPTION (provided by applicant): Mutations that arise after fertilization in somatic cell lineages are linked to cancer and aging an have been shown to contribute to an increasing number of human disorders. Similarly, de novo germline mutations in neuronal genes are responsible for cases of autism, schizophrenia and intellectual disability, suggesting that similar types of somatic mutations could contribute to these and other neurological disorders by providing large-effect mutations in specific cell types, that may act alone or in concert with inherited variants. Despite the growing awareness of the importance of genomic mosaicism for human health, our present understanding of somatic mutation in different cellular lineages of the body and brain is minimal. This question has been difficult to address because conventional genome-wide methods cannot detect variants that are rare within a cell population, and most tissues are composed of diverse cell types and intermixed lineages. Single cell sequencing offers one solution, however, current methods suffer from high error-rates and low resolution, and do not allow for independent validation of mutations detected in merely one cell. A second means to amplify genomes from single cells is through clonal expansion. This is feasible for cancer and some self-renewing cell types, but not for many interesting or aged cell types such as post-mitotic neurons. Here, we propose to use two innovative strategies to profile genomes from individual neurons and control fibroblasts from young and aged mice. First, we take advantage of the only known method to amplify neuronal cells without use of oncogenic factors: cloning by somatic cell nuclear transfer. This will enable deep whole genome sequencing and comprehensive mutational profiling of neuron-derived cell lines. Second, we will use nuclear transfer to produce pairs of sister cells derived from single neurons after one division. Single cell sequencing of replicate sister cells will enable sensitive and accurate detection of de novo copy number variation in a context where bona-fide mutations can be clearly distinguished from DNA amplification artifacts. Our application of these complementary technologies will reveal the full spectrum of genome variation that arises in different cell lineages during development and aging, and will help resolve longstanding hypotheses regarding the extent, impact and origins of neuronal genome diversity.

The human brain contains diverse neural cell types that are differentially responsible for distinct aspects of human behavior, cognition and neurologic disease. Advances in DNA sequencing are providing new insight into human specific neurobiology by providing lists of genes that increase risk for disease or correlate with behaviors and cognitive properties that differ between individuals. However, the lack of available human neuronal subtypes and our limited understanding of which genes are expressed in different human neurons are major barriers to exploiting these growing genomic resources. One way to overcome this barrier is to use direct reprogramming to produce induced neuronal cells in vitro, by transiently expressing transcription factors (TFs) in fibroblasts. Direct reprogramming produces induced neurons that share many features with endogenous neurons, including characteristic morphologies, ligand-evoked synaptic responses and characteristic patterns of gene expression. Reprogramming therefore offers a new tool to identify transcriptional circuits that establish distinct features of neuronal identity. We, and others, have used candidate gene approaches to engineer induced neurons that functionally mimic well-characterized neuronal subtypes, such as the peripheral sensory neurons that detect pain and itch produced recently by my laboratory. These studies led us to hypothesize that direct reprogramming engages conserved transcriptional circuits similar to those that actively maintain neuronal subtype identity in endogenous neurons. This hypothesis predicts that it should be possible to identify multiple TF combinations that induce distinct features of different neuronal subtypes. To test this hypothesis and establish a systematic method to produce and classify human neuronal subtypes, we will conduct an unbiased screen for new TF combinations that can induce human neuronal subtypes in vitro. We will then characterize the induced neurons transcriptionally, morphologically and functionally. We are well suited to perform this study because we recently conducted a pilot screen of ~600 TF pairs and identified more than 70 new pairs that produce candidate induced neurons from mouse fibroblasts. Gene expression profiling and functional analyses of these cells confirm that they exhibit extensive subtype diversity. Therefore, by using unbiased screens to define sets of human TFs that can induce neuronal identity in fibroblasts, we will identify new methods to produce human neuronal cell types with defined functional properties in vitro and establish a database of transcriptional programs and cellular properties that emerge from transient expression of different sets of TFs. These studies will have impact on our understanding of the basic biology of human neuronal diversity and will provide conceptual and practical tools to enable neuroscience researchers to produce diverse subtypes of human induced neurons for research and translational applications.