Here, we propose to elucidate the role of candidate lincRNAs during mammalian brain development using a unique resource of 20 mouse lincRNA knockout models that we have recently generated. We specifically focused on candidates we hypothesized, through previous studies, to play a role in neuronal development. Indeed 15 of the 20 candidates exhibit cell-type and brain specific expression patterns. More importantly, we have already observed lethality phenotypes and gross anatomical abnormalities during brain development in selected mutants.
We aim to explore the molecular and physiological underpinnings of the observed phenotypes as well as of others that we may encounter through this work. We propose to take a comprehensive and systematic approach to address the following questions: What are the spatial temporal dynamics of lincRNA expression in vivo during neuronal development and what gene pathways are regulated? (Aim 1). We will address these questions by leveraging a knock-in lacZ reporter to monitor lincRNA expression patterns and dynamics throughout brain development. We will address the second question through next generation RNA-sequencing studies between wild-type and knockout strains to identify specific genes and pathways regulated by lincRNAs in vivo. What are the physiological and molecular underpinnings of lethality and brain development phenotypes observed in selected lincRNA mutants? (Aim 2). Here, we will address this question through a battery of in vivo experiments to define perturbations to normal neuronal cell fate specification in selected mutants. We will focus first on three lincRNAs that have already shown brain specific phenotypes resulting in lethality and abnormalities of neurogenesis and one expressed in neuronal precursor cells. If time and resources allow we will expand these phenotypic studies to additional strains. How are lincRNAs working on a molecular and mechanistic level to ensure proper neuronal cell fate specification and viability? (Aim 3). Here, we use multifaceted and novel experimental protocols to define the specific regions of lincRNAs required to recover phenotypes. Moreover, we will identify lincRNA protein partners and how they interact to modulate cell fate decisions. The proposed research is driven by our vision, which seeks to unify experimental and computational genomics to push new frontiers in understanding the physiological and molecular regulatory roles of lincRNAs during neuronal development. Collectively, these studies will present the first phenotyping of lincRNA functional roles during neuronal development and will explore their biochemical mechanisms in vivo.

Public Health Relevance

Our research aims to understand a universal problem in human health: How does the same genome present in every cell take on alternate identities that orchestrate distinctive cell states and how are these states misregulated in diseases. We recently discovered a novel class of large intergenic non- coding RNAs (lincRNAs) that have the ability to regulate cellular identity. Here we aim to understand the in vivo relevance for lincRNAs in neuronal development. The ultimate goal of these innovative experimental and computational approaches is to develop novel RNA based therapeutics.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
1R01MH102416-01
Application #
8612093
Study Section
Molecular Neurogenetics Study Section (MNG)
Program Officer
Panchision, David M
Project Start
2014-01-01
Project End
2018-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
1
Fiscal Year
2014
Total Cost
$422,500
Indirect Cost
$172,500
Name
Harvard University
Department
Anatomy/Cell Biology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138