Visualizing endoderm cell populations and their dynamics in the mouse embryo with a Hex-tdTomato reporter

Abstract

Live imaging is the requisite tool for studying cell behaviors driving embryonic development and tissue formation. Genetically encoded reporters expressed under cell type-specific cis-regulatory elements that drive fluorescent protein expression at sufficient levels for visualization in living specimens have become indispensable for these studies. Increasingly dual-color (red-green) imaging is used for studying the coordinate behaviors of two cell populations of interest, identifying and characterizing subsets within broader cell populations or subcellular features. Many reporters have been generated using green fluorescent protein (GFP) due to its brightness and developmental neutrality. To compliment the large cohort of available GFP reporters that label cellular populations in early mouse embryos, we have generated a red fluorescent protein (RFP)-based transgenic reporter using the red fluorescent tdTomato protein driven by cis-regulatory elements from the mouse Hex locus. The Hex-tdTomato reporter predominantly labels endodermal cells. It is a bright RFP-based reporter of the distal visceral endoderm (DVE)/anterior visceral endoderm (AVE), a migratory population within the early post-implantation embryo. It also labels cells of the definitive endoderm (DE), which emerges at gastrulation. Dual-color visualization of these different early endodermal populations will provide a detailed understanding of the cellular behaviors driving key morphogenetic events involving the endoderm.

Keywords: AVE; Definitive endoderm; Gastrulation; Hex; Live imaging; Red fluorescent protein; Visceral endoderm.

Fig. 1.

Hex-tdTomato construct design and validation in cells and early mouse embryo. (A) Schematic representations of the previously published Hex-GFP construct (Rodriguez et al., 2001) and Hex-tdTomato construct depicting the cis-regulatory elements and the inserted fluorescent protein cassette used to generate the Hex-tdTomato transgenic mouse line. (B) Validation of the expression of the Hex-tdTomato construct in two endoderm derived cell lines, Caco2 and Hepa 1-6. Tomato expression can be seen in both cell lines. (C) Expression of Tomato can be detected in Gata6-positive primitive endoderm cells at the late blastocyst stage (E4.5). Note, cytoplasmic Tomato expression highlights primitive endoderm cells extending filopodia (white arrowheads). Panels show 3D renderings of laser confocal images of a whole mount view immunostained for Tomato, Gata6 and Hoechst (DNA), and zoomed-in views of the ICM depicting, Hoechst (DNA), Tomato and Gata6 3-channel merge, Tomato and Gata6 expression 2-channel merge and Tomato and Gata6 single channel images. (D) Expression of Tomato at early post-implantation stages E5.5 and E6.5. Right panels show 3D renderings of laser confocal images of Tomato reporter and F-Actin expression, center panels show Tomato expression only, left panels show 3D surface rendering of Tomato expression counterstained with F-Actin. Yellow arrowheads point to leading cells expressing elevated levels of fluorescence. Blue arrowheads point to rosette-like cell arrangements in the embryonic VE. ADE, anterior definitive endoderm; A, anterior; AVE, anterior visceral endoderm; D, distal; P, posterior; Pr, proximal. Asterisk marks the border of extra-embryonic and embryonic visceral endoderm. Scale bars: (C) 10 µm; (D) 20 µm (E5.5); 20 µm (E5.75); 50 µm (E6.5).

Fig. 2.

Hex-tdTomato is expressed in cells of the embryonic visceral endoderm and definitive endoderm at E7.5. (A-D′) Lateral (A), anterior (B), posterior (C) and ventral (D) whole mount views of an E7.5 Hex-tdTomato embryo. Panels on the left (A-D) show 3D renderings of laser confocal images of Tomato reporter and F-Actin expression and on the right (A′-D′) Tomato expression only. (E,F) Transverse sections through the embryonic part of an E7.5 Hex-tdTomato embryo showing expression of Tomato specific to endodermal cells. (E′,F′) High magnification images of squares in E and F. Arrowhead points to expression in definitive endoderm cells. end, endoderm; epi, epiblast; mes, mesoderm; ml, midline; n, node; ps, primitive streak. Scale bars: (A-D′) 100 µm; (E-F′) 50 µm.

Fig. 3.

Expression of Hex-tdTomato at E8.5 and E9.5. (A-C′) Lateral (A), anterior (B) and posterior (C) whole mount views of an E7.5 embryo. (A-C) 3D renderings of laser scanning confocal images of tdTomato reporter co-stained with F-Actin. (A′-C′) Tomato expression only. (D,D′) Whole mount bright field and red fluorescent views of an E9.5 Hex-tdTomato embryo. (E-G′) Laser confocal images of transverse sections through an E9.5 Hex-tdTomato embryo showing Tomato expression co-stained with F-Actin (E-G) and (E′-G′) Tomato expression only. bi, blood islands; D, dorsal; da, dorsal aorta; L, left; hg, hindgut; isv, intersomitic vessels; nc, notochord; nt, neural tube; pa, pharyngeal arch; paa, pharyngeal arch artery; ps, primitive streak; R, right; uv, umbilical vein; V, ventral. Scale bars: (A-C) 100 µm; (E-G) 50 µm.

Fig. 4.

Correlation in Hex-tdTomato and Hex-GFP reporter expression. (A-H′′) 3D renderings of laser scanning confocal images of whole mount views of E5.5 (A,B′′), E6.5 (C,D′′), E7.5 (E,F′′) and E8.5 (G,H′′) Hex-tdTomato; Hex-GFP embryos expressing Tomato and GFP. (A-H) Panels show Tomato and GFP expression. (A′-H′) Panels show Tomato expression. (A″-H″) Panels show GFP expression. A, anterior; AVE, anterior visceral endoderm; D, distal; DVE, distal visceral endoderm; ml, midline; P, posterior; Pr, proximal. Scale bars: (A,B) 20 µm; (C,D) 50 µm; (E-H) 100 µm.

Fig. 5.

Hex-tdTomato reporter labels a subpopulation of Pdgfra^H2BGFP-positive VE cells, as well as nascent DE and midline cells. (A-F″) 3D renderings of laser scanning confocal images of whole mount views of E5.5 (A-A″), E5.75 (B-B″), E6.5 (C-D″) and E7.5 (E-F″) Hex-tdTomato; Pdgfra^H2BGFP embryos expressing Tomato and GFP. (A′-F′) Tomato expression and (A″-F″) GFP expression. Asterisks mark the border of extra-embryonic and embryonic visceral endoderm. A, anterior; AVE, anterior visceral endoderm; D, distal; DVE, distal visceral endoderm; ml, midline; P, posterior; Pr, proximal. Scale bars: (A,B) 20 µm; (C,D) 50 µm; (E,F) 100 µm.

Fig. 6.

Hex-tdTomato marks a subset of AFP-GFP-positive VE cells. 3D renderings of whole mount views of (A-B′′) E5.5, (C-D′′) E6.5, (E-F′′) E7.5 and (G-H′′) E8.5 Hex-tdTomato; AFP-GFP embryos. Panels show expression of (A-H) Tomato and GFP, (A′-H′) Tomato and (A″-H″) GFP. A, anterior; AVE, anterior visceral endoderm; D, distal; DVE, distal visceral endoderm; L, left; ml, midline; P, posterior; Pr, proximal; R, right. Scale bars: (A,B) 20 µm; (C,D) 50 µm; (E-H) 100 µm.

Fig. 7.

Live imaging a Hex-tdTomato; Hex-GFP double transgenic dual reporter embryo reveals cellular behaviors associated with migration of DVE/AVE cells. (A-H) Representative time frames taken from a 3D time lapse imaging experiment of a E5.5 Hex-tdTomato; Hex-GFP over a period of 660 min. The embryo was imaged every 10 min. The series depicts the initiation of AVE migration. Tomato- and GFP-positive cells can be seen to migrate towards the future anterior of the embryo. (A-H) Panels show merge of red and green channels with bright field image of whole mount view of embryo. (A) White line outlines the contour of the embryo. Note, white arrows point to anteriorly oriented protrusions of AVE cells. (A′-H‴) Zoomed in views of the distal part of the embryo, corresponding to the region of the VE overlying the epiblast. (A′-H′) Tomato and GFP fluorescence. (A″-H″) Tomato fluorescence. (A‴-H‴) GFP fluorescence. Asterisks mark the border of extra-embryonic and embryonic visceral endoderm. A, anterior; D, distal; DVE, distal visceral endoderm; P, posterior; Pr, proximal; Scale bar: 20 µm.