Fragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health (DC13074 and DC17267 to Y.W.), the United States – Israel Binational Science Foundation (2015087 to Y.W. and D.S.-D.), the Israel Science Foundation (1515/16 to D.S.-D.) and the Office of the Chief Scientist, Ministry of Health (3-0000-33793 to D.S.-D.). Deposited in PMC for release after 12 months.
We thank Edwin W Rubel (University of Washington) for facilitating the collaboration between Y.W. and D.S.-D. We are grateful to David Fradkin for assistance with the IncuCyte system, Itzhak Sibony and Tom Schultheiss for the Lhx2/9 antibody, and Daniel Lazar for help with the tissue culture. We thank Jeniffer Darnell (Rockefeller University) for the hFMRP-EGFP plasmid.
© 2020. Published by The Company of Biologists Ltd
- Auditory circuit
- Autism spectrum disorder
- Axon development
- Axon fasciculation
- Axon targeting
- Fragile X syndrome
- RNA-binding proteins