Visualization of lymphatic vessels by Prox1-promoter directed GFP reporter in a bacterial artificial chromosome-based transgenic mouse

Abstract

Although the blood vessel-specific fluorescent transgenic mouse has been an excellent tool to study vasculogenesis and angiogenesis, a lymphatic-specific fluorescent mouse model has not been established to date. Here we report a transgenic animal model that expresses the green fluorescent protein under the promoter of Prox1, a master control gene in lymphatic development. Generated using an approximately 200-kb-long bacterial artificial chromosome harboring the entire Prox1 gene, this Prox1-green fluorescent protein mouse was found to faithfully recapitulate the expression pattern of the Prox1 gene in lymphatic endothelial cells and other Prox1-expressing organs, and enabled us to conveniently visualize detailed structure and morphology of lymphatic vessels and networks throughout development. Our data demonstrate that this novel transgenic mouse can be extremely useful for detection, imaging, and isolation of lymphatic vessels and monitoring wound-associated lymphangiogenesis. Together, this Prox1-green fluorescent protein transgenic mouse will be a great tool for the lymphatic research.

Figure 1

GFP expression pattern in the Prox1-GFP BAC transgenic mice. (A) GFP expression in embryos and newborn mice: stereoscopic images of a Prox1-GFP (left) and a wild-type embryo (right) (E14.5) in the bright-field and green fluorescence channel (i-ii). (iii-v) A patch of Prox1-GFP embryonic back skin was peeled to visualize the dermal lymphatic network (v, enlarged image of the boxed area of subpanel iv). (vi) Leading front (arrows) of embryonic dermal lymphatic vessels. (vii-viii) Mesenteric lymphatic vessels and lymphatic valves (arrows) in a newborn mouse (P1). Bright-field and green fluorescent images of the embryonic liver (ix-x), the pancreas (xi-xii), the heart (xiii-xiv), the nervous system in the tail (xv-xvi), the eye (xvii-xviii), isolated eyeball (xix-xx), and the brain (xxi-xxii). Expression of GFP reporter in embryonic neural tube (xxiii), hepatocytes (xxiv), heart muscle (xxv), and intestine (xxvi) of newborn mice. (B) GFP expression in the Prox1-GFP BAC transgenic adult mice: bright-field and green fluorescent imaging allows a convenient distinction of transgenic (left) versus wild-type control (right) mice (i-ii). Cross sections of the eye lens (iii) and the retina (iv). Lymphatic vessels are shown in whole-mount preparations of the trachea (v), inner side of the skin (vi), ear edge (vii), Peyer patch (viii), mesentery (ix), surface of intestine (x) and lymph nodes (xi), diaphragm (xii-xiii), and an enlarged view (xiv) of boxed area in panel xiii. (xv-xvi) The ear of a Prox1-GFP mouse was wounded with an ear punch. *Lymphatic vessel regeneration was visualized at the edge of the wound at day 7. Filopodia of sprouting lymphatic endothelial cells are shown in a boxed area in panel xv and marked with arrows in the enlarged image (xvi). The thoracic duct of a Prox1-GFP mouse can be easily visualized in the retroperitoneal space (xvii) with an enlarged view (xviii). An isolated thoracic duct clearly shows a strong GFP positivity (xix-xx).

Figure 2

Colocalization of the GFP signal with various lymphatic markers throughout development. Whole-mount staining of dermal lymphatics in Prox1-GFP transgenic embryos (E14.5) against CD31 (A-C), Prox1 (D-F), and LYVE1 (G-I) show that GFP expression is colocalized with the expression of Prox1 and LYVE1. Moreover, staining of skin cross section of newborn pup (P1) against CD31 (J-L) and podoplanin (PDPN, M-O) specifically found GFP signal in lymphatic endothelial cells, suggesting a lymphatic-specific expression of GFP. (P-R) *Podoplanin staining of a cross section of Prox1-GFP embryo at E10.5 shows the budding LECs (arrows) from the embryonic cardinal vein. Scale bars represent 100 μm with an exception (G-I, 50 μm).