A bright single-cell resolution live imaging reporter of Notch signaling in the mouse

Abstract

BackgroundLive imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.ResultsTo generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.ConclusionBy using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport

“…To further monitor Notch activity in intestinal lacteals, we analyzed CBF1:H2B-Venus mice, where CBF1 binding sites drive expression of histone H2B-Venus (49). In agreement with DLL4 expression patterns and consistent with the previously described Notch activity in these vessels, arterioles were highly Venus + , while venule BECs, stained for mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), expressed lower levels or no transgene ( Figure 4D and refs.…”

“…fl/fl , Pdgfb-CreERT2, Prox1-CreERT2, Rosa26-EYFP, and CBF1:H2B-Venus mice were previously described (27,49,(75)(76)(77) ing reflects a need for continuous lacteal regeneration due to the unique small intestine microenvironment, which itself is constantly renewing. Mechanistically, we show that DLL4/Notch signaling, acting downstream of VEGFR3 and VEGFR2, mediates part of such a maintenance and regeneration program in lacteals.…”

Single-cell analysis reveals lineage segregation in early post-implantation mouse embryos

“…Briefly, embryos containing only the EPI, VE, and EXE were dissociated into single-cell suspension after the PE and EPC were removed with digestion. mRNA of each cell was reversetranscribed and amplified to obtain cDNA, and the expression of the EPI marker Oct4 (30 -34), the VE marker Gata6 (35)(36)(37), and the EXE marker Hand1 (38,39) Principal component analysis (PCA) on transcriptome data of 124 samples was conducted to visualize the relationship among distinct cell types in the embryo. As anticipated, data clustered according to the expression of Oct4, Gata6, or Hand1 (Fig.…”

NOTCH activation interferes with cell fate specification in the gastrulating mouse embryo

“…Indeed, NOTCH signalling is likely to be active in early post-implantation embryos as suggested by the expression of the CBF:H2B-Venus NOTCH reporter and by the immunodetection of cleaved NICD in post-implantation embryos (Del Monte et al, 2007;Nowotschin et al, 2013). Moreover, in vitro studies on mouse (mESC) and human (hESC) embryonic stem cells support a role for NOTCH signalling in shaping early cell fate decisions.…”