043-CO-2-Microcine | Characterizing multiple states of neuronal development in the adult hippocampus using single-nuclei RNA-seq

Cellular and Molecular Neurobiology

Author: Natali Rasetto | Email: rasettonatali@gmail.com

Natalí Belén Rasetto , Damiana Giacomini , Ariel Berardino , Tomás Vega Waichman , Maximiliano Beckel , Daniela Di Bella , Juliana Brown , Paola Arlotta , Ariel Chernomoretz , Alejandro Schinder

1° Laboratory of Neuronal Plasticity, Leloir Institute-CONICET, Buenos Aires, Argentina.
2° Laboratory of Integrative Systems Biology, Leloir Institute-CONICET, Buenos Aires, Argentina
3° Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA.

Adult hippocampal neurogenesis plays a critical role in spatial memory formation, context discrimination, and clearance of memory traces. In the mouse dentate gyrus, the maturation of adult-born granule cells lasts several weeks and can be divided in 4 phases based on electrophysiological and morphological features. However, the molecular mechanisms underlying the progression through those discrete phases are still unknown. We have proposed that maturation is driven by sequential changes in the gene expression program, and should be revealed by transcriptome analysis. We thus set up an approach for high-throughput single-nuclei RNA sequencing applying Chromium 10X Genomics technology to interrogate the transcriptomic profile of new granule cells at different ages. We used Ascl1CreERT2;CAGfloxStopSun1sfGFP mice to allow conditional expression of Sun-1/sfGFP in the nuclear membrane of developing granule cells at identified ages and isolate fluorescent nuclei using FACS. Clusterization of two separate datasets identified multiple partitions that define a pathway from radial glia-like cells to mature neurons. Several clusters represent intermediate stages of maturation that were previously unknown. The emergence of novel transcriptional markers for the intermediate states was validated by in situ hybridization using RNAscope. These results are beginning to reveal key players involved in neuronal maturation and function with high temporal resolution.