Tg(Afp-GFP) expression marks primitive and definitive endoderm lineages during mouse development

Abstract

Alpha-fetoprotein (Afp) is the most abundant serum protein in the developing embryo. It is secreted by the visceral endoderm, its derivative yolk sac endoderm, fetal liver hepatocytes, and the developing gut epithelium. The abundance of this protein suggested that Afp gene regulatory elements might serve to effectively drive reporter gene expression in developing endodermal tissues. To this end, we generated transgenic mouse lines Tg(Afp-GFP) using an Afp promoter/enhancer to drive expression of green fluorescent protein (GFP). Bright GFP fluorescence allowed the visualization, in real time, of visceral endoderm, yolk sac endoderm, fetal liver hepatocytes, and the epithelium of the gut and pancreas. Comparison of the localization of green fluorescence with that of endogenous Afp transcripts and protein indicated that the regulatory elements used to generate these mouse lines directed transgene expression in what appeared to be all Afp-expressing cells of the embryo, but only in a subset of fetal liver cells. The bright GFP signal permitted flow cytometric analysis of fetal liver hepatocytes. These mice represent a valuable resource for live imaging as well as identification, quantitation, and isolation of cells from the primitive and definitive endoderm lineages of the developing mouse embryo.

Figure 1. Onset of Afp-GFP transgene expression

(A) Schematic representation of the Afp-GFP transgenes used in this study. (B) GFP fluorescence was not detected at preimplantation stages including late (E4.5) transgenic blastocysts. Brightfield image of E4.5 embryos with implantation sites (arrowhead). Individual time points from a confocal 3D (z-stack) timelapse sequence of Afp-GFP embryos (C–M) depicting the onset of detectable transgene expression and dynamic morphology of fluorescent cells. Rendered confocal data overlayed onto brightfield images of blastocyst outgrowths at t = 12 + 0 hr, referring to 12 hr after plating and the start of the time-lapse imaging sequence (C), two outgrowths contain cohorts of cells that express GFP (white arrowheads). The onset of GFP expression in a third embryo, less advanced outgrowth is detected after 1 hr of culture (D, red arrowhead). De novo initiation of GFP expression in a second cell at a second site is observed within the same outgrowth (yellow arrowhead) after 2 hr in culture (E). High magnification rendered confocal images from a timelapse sequence of an Afp-GFP outgrowth imaged over 90 min (F–M), depicting a cell division and dynamic, protrusive cell morphology (F–I, fluorescence overlayed on bright field; J–M, fluorescence channel at a higher magnification). (F) At t = 12 + 0 min, two cells express GFP (white and red arrowheads). (G,H) One cell has divided (red arrowhead), resulting in a third GFP expressing cell (yellow arrowhead). (J) At t = 12 + 0 min, one GFP expressing cell (white arrow) has extended a dynamic, unidirectional projection (white arrowhead). Thereafter, a second GFP expressing cell (K, red arrow) extended a unilateral projection along the existing projection (red arrowhead), at which point the projection from the first cell was retracted (L, white arrowhead) and subsequently projected in a different direction (M, white arrowhead). Scale bars represent 50 μm.

Figure 2. Visualization of GFP fluorescence in early postimplantation stage Afp-GFP embryos

Fluorescence was restricted to the distal visceral endoderm (primarily the cells overlying the epiblast) in prestreak (E5.5) and early streak (E6.5) stage embryos. Brightfield (A,E), single confocal sections taken from a z-stack (B,F), merge of confocal and bright field channels (C,G), and 3D projection of the z-stack of confocal images (D,H). Panels I and J show projections of embryos shown in A–D and E–H, respectively. Embryos have been counterstained with DRAQ5 to highlight nuclei. Depicted images are of red (DRAQ5) and green (GFP) fluorescent channel merges. (I,J) Rendered 2-channel images were rotated 90 degrees around the proximo-distal axis of the embryo, revealing the three dimensionality of GFP localization in the context of embryo morphology. Scale bar represents 50 μM.

Figure 3. GFP expression parallels Afp mRNA localization by the headfold to somitogenesis stages and is restricted to the yolk sac endoderm

Brightfield (A) and widefield fluorescent (B) images of a headfold stage Afp-GFP embryo and (C) a stage matched embryo depicting Afp mRNA localization. Brightfield (D) and widefield fluorescent (E) images of a 5 somite stage Afp-GFP embryo and (F) a stage matched embryo depicting Afp mRNA localization. Brightfield (G) and widefield fluorescent (H) images of a 11 somite stage Afp-GFP embryo and and (I) a stage matched embryo depicting Afp mRNA localization. (J–L) Data from a confocal z-stack of E8.5 yolk sac counterstained with DRAQ5 to highlight nuclei (red). Green fluorescence is localized to the yolk sac endoderm (white arrowheads) and excluded from yolk sac mesoderm (blue arrowheads). (J), GFP channel (depicting orthogonal views of single xy, xz and zy slices); (K), GFP channel merged with DRAQ5 channel (depicting orthogonal views of single xy, xz and zy slices; (L), 3D rendered z-stack rotated 45 degrees. Abbreviations: hf, headfold; 5 som, 5 somite; 11 som, 11 somite. Scale bar represents 50 μM.

Figure 4. By midgestation, GFP is localized to the yolk sac and definitive endodermal derivatives

Widefield fluorescence overlayed on brightfield images demonstrates that GFP is restricted to the yolk sac (A,I) and liver (B,J) of Afp-GFP E9.5 and E11.5 embryos. Rendered z-stacks of confocal sections acquired on vibratome sections through the liver region at E9.5 (C,D) and E11.5 (K). The approximate planes of transverse section are depicted by dashed red lines in panels B and J. (L) High magnification rendered z-stack of the fetal liver at E11.5 reveals pseudopodial-like projections (white arrowheads) observed on hepatocytes. (M) Color-coded depth projection depicting a three dimensional data set in two dimensions, the depth being represented by color, of a rendered confocal image of the z-stack of the first time point (t = 0 h) from a time-lapse imaging sequence of a vibratome section of a fresh E11.5 liver in slice culture (dark blue represents 0 μm; red represents 50 μm). (N) At t = 1 hr, a liver cell extends a unilateral projection in one direction (white arrowhead). (O) At t = 2 hr, the same cell extends a second projection in a different direction (red arrowhead). (P) At t = 3 hr, the projections show dynamic extension and retraction (white and red arrowheads). In situ hybridization of Afp RNA expression in stage matched embryos reveals localization to the yolk sac (E,Q) and liver (F,R). Vibratome section in situ hybridization of Afp RNA expression in liver at E9.5 (G,H) and E11.5 (S,T). Scale bars represent 100 μm (C,G,K,S); 50 μm (D,H,L,M,N,T).

Figure 5. Expression of the Afp-GFP transgene in the liver of E13.5 and E14.5 embryos

(A) Expression of the Afp-GFP transgene in the E13.5 liver (right). A liver from a wild type littermate is shown (left). Images are overlays of brightfield and GFP channel fluorescence. (B) Immunostaining of E14.5 fetal liver cytospins. All GFP(+) cells express endogenous Afp protein (red; more than 80 GFP(+) cells examined). (C) GFP(+) cells also expressed the endodermal protein Hnf4α (left panel; more than 200 GFP(+) cells examined) but did not express hematopoietic markers (middle and right panels). Immunostaining of cryosections revealed Ter-119(+) erythroid (middle panel) and F4/80(+) macrophage (right panel) populations within the fetal liver. GFP-expressing cells are interspersed with these cells. The characteristic hepatoblastic morphology of the GFP(+) cells is evident at high magnification (right panel). (D,E) Flow cytometric analysis of cells from E14.5 Afp-GFP fetal livers. (D) Contour plot of GFP fluorescence versus forward scatter (FSC, reflecting cell size). The GFP(+) cells were large, as evidenced by their high FSC. (E) The GFP(+) population (9% of total) was gated and examined for expression of surface antigens. Numbers above the bars represent the percentage of GFP(+) cells expressing the indicated surface antigen. The X axis indicates relative logarithmic fluorescence units.

Figure 6. Expression of GFP in endoderm-derived tissues of late stage embryos

Strong expression of GFP was observed in the yolk sac of E16.5 embryos (A, bright field; B, GFP; C, composite). Bright expression was also detected in the fetal liver (D, composite of bright field and GFP channel fluorescence), intestine (E, orange arrow), pancreas (E, white arrow), and (ectopically) in the brain (F, composite). (G) Yolk sac expression of GFP was restricted to the endodermal layer (E14.5 embryo). Staining for Ter-119, an erythroid marker, highlights erythroblasts within blood vessels adjacent to the GFP(+) visceral endoderm. (H) GFP(+) endodermal cells express E-cadherin in the yolk sac. Note that E-cadherin is expressed at cell-cell junctions while GFP is found within the cytoplasm. GFP(+) cells in the developing intestine (I) and pancreas (J) expressed the epithelial marker E-cadherin.