Alexander A. Pollen - US grants

DESCRIPTION (provided by applicant): The evolutionary expansion of the human neocortex required increased production of excitatory and inhibitory neurons. The balance between excitation and inhibition is critical for normal neurological function, and yet we know little about the human-specific genetic changes underlying increased neurogenesis. The recent availability of complete genome sequence from humans and a diverse range of mammals makes large scale sequence comparisons possible. Regulatory elements that are conserved in other mammals, but surprisingly missing in humans could contribute to human specific biology. Initial work on the project shows that humans have lost a forebrain specific regulatory element in zones of inhibitory interneuron production near the tumor suppressor gene GADD45g, which is specifically expressed in neural progenitors during development. GADD45g represses cell cycle in culture and GADD45g down-regulation is strongly associated with pituitary adenomas, yet the function of GADD45g in nervous system development has not yet been explored. This project will characterize in detail the neuronal lineages affected by the human specific genetic change using genetic labeling techniques in mouse stable lines expressing Cre-recombinase driven by the enhancer missing in humans. To put the mouse expression data in the context of changes in primate gene regulation, this project will also identify the upstream regulators of the DNA sequence that is missing in humans. Finally, this project will characterize how loss of the GADD45g gene affects brain development in mouse using an existing knockout line not previously analyzed for nervous system phenotypes. Collectively, these experiments will provide an in depth study of how a human- specific genetic change may contribute to specialized aspects of human brain development. PUBLIC HEALTH RELEVANCE: This research is relevant to public health because GADD45g mis-regulation is strongly associated with multiple cancers including pituitary adenomas, a major form of brain cancer. Additionally, the balance between neuronal excitation and inhibition is critical for normal neurological function and may be disrupted in disorders as diverse as epilepsy, Down's syndrome and autism. Identifying how human specific genetic changes regulating GADD45g expression affect the production of inhibitory interneurons may both aid in understanding the role of GADD45g in human brain tumors, and in understanding uniquely human aspects of inhibitory neuron production.

Project summary/abstract Genetic changes over the last six million years have transformed the structure and function of the human brain. However, identifying the key genetic changes that transform developmental process has been challenging because developmental tissue is largely inaccessible to comparative and functional studies. This project will establish a systematic quantitative genetic approach for discovering the human-specific genomic changes that underlie unique features or vulnerabilities of the developing human brain. Our project leverages human and great ape pluripotent stem cell and cerebral organoid models that we have established to perform comparative analysis of gene activity and cell behavior during hominid cortical development. In addition, by incorporating advances in genome engineering screens and novel chromosome engineering strategies, this project will perform large-scale functional analysis of human-specific variants in the context of key processes of cortical development. Finally, this project involves the development of novel and generalizable tools for both the derivation of specific cell types from pluripotent stem cells across many individuals and species and for high-throughput analysis of cellular phenotypes. Ultimately, our vision of success will be a platform that enables a quantitative understanding of the extent to which individual human-specific mutations explain variation in specialized aspects of human brain development.