The long-term goals of the proposed research are both to elucidate central molecular controls over development and diversity of neocortical callosal projection neuron (CPN) connectivity, and to identify potential causes and therapeutic approaches to disease involving CPN circuitry. CPN are the broad population of inter-hemispheric pyramidal neurons whose axons connect the two cerebral hemispheres via the corpus callosum. CPN play key roles in high-level associative, integrative, cognitive, behavioral, sensory, and motor functions, based on precise, area-specific CPN subtype connectivity and diversity. Disruptions in CPN development are correlated with deficits in multiple disorders, including agenesis of the corpus callosum, autism spectrum disorders, and schizophrenia. Currently, how the remarkable diversity of CPN subtypes and connectivity is specified, and how transcriptional programs implement specific connectivity via local, cell-autonomous effectors, is unknown. Our lab recently identified a combinatorially-expressed set of genes that both define CPN as a broad population, and identify novel subpopulations of CPN during development (Neuron, 2005, 2016a,b; J Neurosci, 2009; Cer Cor, 2016a,b). We also developed innovative approaches to investigate subtype-specific, subcellular growth cone (GC) molecular machinery. Building on this work, we propose deep and rigorous functional investigation of Cited2 control over precise CPN connectivity & circuit wiring, including RNAs & proteins detected uniquely in GCs. Cited2 is an exemplar transcriptional co-regulator that we hypothesize functions importantly in development of precise areally- and functionally-specific CPN circuitry in somatosensory cortex, and its dysfunction elucidates disorders of CPN connectivity and diversity. We have already identified that Cited2 regulates and refines two stages of precise CPN development and diversity, functioning 1) broadly in basal progenitors to regulate generation of superficial layer CPN, and 2) postmitotically in an area-restricted manner to refine distinct, precise identity and development of somatosensory (S1) CPN. To connect Cited2 transcriptional regulation to local implementation of S1 CPN connectivity in developing GCs, we propose to:
Aim 1) investigate CPN-autonomy of Cited2 regulatory function in S1 CPN postmitotic development and connectivity, via novel mosaic, recombinase-based genetic manipulation technology (?BEAM?) for dual population analysis;
Aim 2) investigate GC & soma RNA & proteomes of WT vs Cited2 cKO S1 CPN during axon development via new and innovative approaches, to gain direct mechanistic understanding of CPN circuit development at critical developmental stages;
Aim 3) investigate the specific function of GC-localized downstream effectors that are dysregulated in Cited2-null CPN;
and Aim 4) investigate the integrated function of precise CPN circuit development in cognitive & ASD-relevant behavior. Together, the proposed studies will provide substantial insight from gene to circuit to behavior into molecular control over development, diversity, and precision of connectivity of CPN subtypes with distinct function and integration of cortical information, processes centrally disrupted in human disorders. Controls over CPN connectivity are now essentially unknown, and transcriptional dysregulation has not been previously connected to downstream local effectors of circuit development. This research will contribute to understanding cortical organization, function, and potentially toward prevention, diagnosis, and therapy of human disorders.

Public Health Relevance

The cerebral cortex, where high-level cognition, association, integration, and motor and sensory processing occurs, contains thousands of distinct types of specialized nerve cells (neurons) enabling it to perform such complex tasks; callosal projection neurons (CPN) are the class of neurons connecting the cerebral hemispheres to integrate cortical information, playing key roles in human behavior and disease. People with abnormalities of their corpus callosum (CC), the structure composed of CPN axons linking the hemispheres, exhibit a range of cognitive and behavioral disease, including intellectual disability, autism, and some types of mental illness, highlighting the importance of proper CPN/CC development; however, little is known about genes and molecules regulating CPN development. Building on recent work identifying the first candidate CPN control molecules, this project will pursue deep and rigorous functional investigation of a newly identified transcriptional co-regulator, Cited2, that appears to centrally regulate development and refinement of this important neuronal population; these investigations will 1) apply novel experimental approaches to identify critical functions of this control gene in development of CPN connections in mice; 2) more broadly elucidate networks of molecules in axon ?growth cones? controlled by Cited2 whose disruption might cause and underlie CPN abnormalities and human diseases involving CPN; and 3) have future potential to identify new ways to diagnose, treat, or prevent such cognitive, associative, and behavioral disorders.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
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Riddle, Robert D
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Harvard University
Anatomy/Cell Biology
Schools of Arts and Sciences
United States
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