TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex

Abstract

Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA>GFPstop>HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the CA>GFPstop>HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.

SLAM-ITseq: Sequencing cell type-specific transcriptomes without cell sorting

“…Then, they pulled-down the thio-RNA using a biochemical isolation method and quantified by RNA-seq to determine enrichment level of labelled RNA over the total RNA. Even though similar methods have been tested in various model organisms ( Erickson and Nicolson, 2015 ; Chatzi et al, 2016 ; Tomorsky et al, 2017 ), technical and analytical challenges limit their application. First, the biochemical isolation methods of thio-RNA have been shown to have high background noise, which makes it difficult to distinguish lowly labelled RNA from the background noise.…”

Precise levels of Nectin-3 and an interaction with Afadin are required for proper synapse formation in postnatal visual cortex

“…The expression patterns of nectin-1 and nectin-3 were assayed by nonradioactive colorimetric RNA in situ hybridization, using solutions and probes as previously described 21,[25][26][27] . Briefly, animals were perfused and brains were fixed overnight in 4% PFA and then cryoprotected in a 30% sucrose solution.…”

“…Here we identify a role for nectin-3 in the development of visual cortical neurons by examining dendritic spine densities after manipulating neuronal nectin-3 expression in vivo. Mammalian cortex has a conserved laminar structure, and nectin-1 and nectin-3 both have distinct upper layer expression patterns in postnatal mouse cortex 16,21,22 . The specific expression of nectin-3 in upper cortical layers may help facilitate the large developmental changes in visual response properties observed in this layer after eye opening relative to other layers (decreased firing rate and increased orientation selectivity) 9 .…”

Uncovering cell type‐specific complexities of gene expression and RNA metabolism by TU‐tagging and EC‐tagging

“…These nucleosides are taken up by all cells and enter RNA biosynthetic pathways independent of UPRT or CD:UPRT. Another advantage of the type II design is that reference RNA is subject to the same purification steps as the TU-tagged/EC-tagged RNA, limiting the chance that a technical bias may be introduced into only one sample (Tomorsky, DeBlander, Kentros, Doe, & Niell, 2017).…”