Afp::mCherry, a red fluorescent transgenic reporter of the mouse visceral endoderm.

Macroscopic changes in tissue or organ structure result from coordinated changes in the arrangements and shapes of cells. To this end, live imaging of genetically-encoded fluorescent protein reporters is increasingly being used to investigate details of the cellular behaviors that underlie the large-scale tissue rearrangements that shape the embryo. The cell movements underlying morphogenesis of the early mouse embryo are complex and not well understood. Gastrulation is the event that results in the formation of the three germ layers from the pluripotent epiblast and leads to the elaboration of the embryonic axes. Prior to the initiation of gastrulation, the mouse embryo comprises a bilaminar cup-shaped structure (often referred to as the egg cylinder stage) consisting of visceral endoderm, an epithelium that encapsulates the extraembryonic ectoderm proximally, and the epiblast distally (Nowotschin and Hadjantonakis, 2010). The proximal visceral endoderm overlying the extraembryonic ectoderm has generally been referred to as the exVE, while the proximally positioned visceral endoderm overlying the epiblast has been referred to as the emVE (Mesnard et al., 2006). The visceral endoderm, which has long been recognized as critical for nutrient uptake and transport, also plays a role in the morphogenesis and patterning of the epiblast (Srinivas, 2006). Using a transgenic strain of mice in which we placed a green fluorescent protein (GFP) reporter under the transcriptional control of the enhancer/promoter sequences from the Alpha-fetoprotein (Afp) gene, we could label the visceral endoderm of the early postimplantation embryo as well as many of its derivatives, including the visceral yolk sac (Kwon et al., 2006). Furthermore, we noted that when compared to other cis-regulatory elements driving visceral endoderm-specific transgene expression, for example from the Transthyretin (Ttr) locus, Afp provided more robust levels of reporter expression (Kwon and Hadjantonakis, 2009). As a consequence we noted the perdurance of GFP protein in cells of the visceral endoderm overlying the epiblast (often referred to as emVE) (Mesnard et al., 2006; Nowotschin and Hadjantonakis, 2010) that would otherwise have downregulated transcription of many visceral endoderm markers including Afp and Ttr (Kwon et al., 2008). The Afp::GFP strain therefore served as a short-term visceral endoderm lineage tracer. 3D time-lapse analyses of the GFP-positive visceral endoderm in Afp::GFP embryos revealed the widespread dispersal of the emVE epithelium during gastrulation. We hypothesized that visceral endoderm dispersal was achieved through widespread intercalation of epiblast cells into the pre-existing visceral endoderm epithelium (Ferrer-Vaquer et al., 2010; Kwon et al., 2008; Nowotschin and Hadjantonakis, 2010). These genetic-labeling and live imaging studies have led to a revised model of mouse gut endoderm morphogenesis, in which epiblast-derived cells egress multi-focally into the emVE epithelial layer and in doing so disperse emVE cells, rather than displacing the VE en masse (Kwon et al., 2008). Furthermore, the observation that in Afp::GFP transgenic embryos GFP-positive emVE cells remained associated with the epiblast, and at later stages they or their descendents were detected within the gut tube, suggested that at least some of the cells comprising the emVE, a lineage previously believed to be exclusively extraembryonic, might contribute to the embryo-proper, thereby challenge the notion of a strict segregation between extraembryonic and embryonic tissue lineages in mammals (Kwon et al., 2008). The majority of mouse fluorescent reporter strains are based on GFP. Originally isolated from the jellyfish Aequorea victoria, this fluorescent protein is the most widely used reporter across model systems. GFP has been extensively engineered and mutated to obtain variants exhibiting improved photostability and brightness, such as EGFP, as well as spectrally-distinct variants that are amenable to use in mice (Nowotschin et al., 2009b). Mouse reporter strains expressing fluorescent colors other than GFP are valuable for co-visualization studies with GFP, where relative positioning and relationship between two different tissues or compartments within cells is being investigated (Nowotschin et al., 2009a). In addition, due to their longer wavelength excitation and emission spectra, red fluorescent proteins are favorable for live imaging studies because of their reduced cellular toxicity. There is therefore a growing need for the development of mouse live imaging reporter strains in colors other than green. We recently generated a Ttr::RFP strain, in which the red fluorescent protein mRFP1 (Campbell et al., 2002) was expressed under the control of the Ttr promoter (Kwon and Hadjantonakis, 2009). In this reporter strain mRFP1 labeled the entire visceral endoderm. However, since comparative reporter experiments suggested that the Ttr cis-regulatory elements do not drive transgene expression as robustly as those from the Afp locus (Kwon and Hadjantonakis, 2009), we wanted to develop spectral-variant reporter strains using the Afp cis-regulatory elements. The weak expression of the Ttr::RFP coupled with the increasing necessity for labeling tissues such as the visceral endoderm in co-visualization experiments with other GFP-based reporter mouse lines led us to generate a new transgenic mouse strain expressing the monomeric red fluorescent protein mCherry (Shaner et al., 2004), under the Afp cis-regulatory elements. Due to its fast maturation, brightness and high photostability, mCherry has already been shown to be a suitable RFP for co-visualization live imaging experiments ex vivo in mouse embryonic stem (ES) cells, and in vivo in both mouse embryo chimeras comprising mCherry expressing ES (Nowotschin et al., 2009a), and in transgenic mice (Fink et al., 2010). Here we report the generation of a transgenic Afp::mCherry mouse strain that expresses the monomeric red fluorescent protein mCherry specifically in the visceral endoderm and its derivatives, the visceral yolk sac and gut endoderm, as well as in the fetal liver and pancreas. In addition, we show the Afp::mCherry reporter exhibits mCherry expression in hepatocytes, as well as in liver, pancreas, digestive tract and brain of postnatal mice. Our analyses confirm that generally the mCherry reporter in the Afp::mCherry strain is expressed in an equivalent spatio-temporal pattern as the GFP reporter in the Afp::GFP strain, with slightly reduced reporter perdurance. By co-visualization we demonstrate the utility of Afp::mCherry reporter as a suitable alternative to the Afp::GFP reporter in experiments where it is necessary to label visceral endoderm cells in a color other than green, thereby making combinatorial reporter visualization studies now possible.