Development and Characterization of A Novel Prox1-EGFP Lymphatic and Schlemm’s Canal Reporter Rat

Abstract

The lymphatic system plays a key role in tissue fluid homeostasis, immune cell trafficking, and fat absorption. We previously reported a bacterial artificial chromosome (BAC)-based lymphatic reporter mouse, where EGFP is expressed under the regulation of the Prox1 promoter. This reporter line has been widely used to conveniently visualize lymphatic vessels and other Prox1-expressing tissues such as Schlemm’s canal. However, mice have a number of experimental limitations due to small body size. By comparison, laboratory rats are larger in size and more closely model the metabolic, physiological, and surgical aspects of humans. Here, we report development of a novel lymphatic reporter rat using the mouse Prox1-EGFP BAC. Despite the species mismatch, the mouse Prox1-EGFP BAC enabled a reliable expression of EGFP in Prox1-expressing cells of the transgenic rats and allowed a convenient visualization of all lymphatic vessels, including those in the central nervous system, and Schlemm’s canal. To demonstrate the utility of this new reporter rat, we studied the contractile properties and valvular functions of mesenteric lymphatics, developed a surgical model for vascularized lymph node transplantation, and confirmed Prox1 expression in venous valves. Together, Prox1-EGFP rat model will contribute to the advancement of lymphatic research as a valuable experimental resource.

Organogenesis and Distribution of the Ocular Lymphatic Vessels in the Anterior Eye: Implication to Glaucoma Surgery Site Selection

“…Thus, the identity, character, and function of this enigmatic lymphatic sac need further investigation. We also confirmed this pattern of nasal-side predilection of the ocular lymphatic development using a different species, Prox1-EGFP lymphatic reporter rats (37). Indeed, we observed a comparable pattern of ocular lymphangiogenesis in rats: the limbal and conjunctival lymphatics in newborn rat pups (P2) migrated from the nasal side and bifurcated to encircle the cornea (Fig.3L).…”

“…As we and others have previously documented (3-6, 37), both the ocular lymphatics and Schlemm's canal were readily detectable in the Prox1 promoter-driven reporter mice by their EGFP expression ( Fig It has not been reported whether the limbal lymphatics harbor the luminal valves, a hallmark of collecting lymphatics, and how they extend beyond the limbus area. Toward these questions, we next performed further studies on the limbal lymphatics using two different rodent species, Prox1-tdTomato mice (36) and Prox1-EGFP transgenic rat (37). Indeed, we found that the limbal lymphatics are directly connected to the conjunctival lymphatics through multiple short lymphatics present along the corneal edge ( Fig.1Q-U).…”

“…We next set out to perform in-depth morphological analyses on the ocular lymphatics using Prox1-tdTomato mice (36) and Prox1-EGFP transgenic rat (37). The entire heads of the reporter animals were fixed with paraformaldehyde to preserve the structures of the ocular and surrounding tissues.…”

“…The eyesfrom Prox1 reporter pups at postnatal (P) days 0.5, 2, 4, 6, 8, and 10, and 6-weeks old adult were enucleated, and cornea flat mounts were prepared. Asterisks (*) point the nasal side of the eye and pound (#) signs label the ocular muscles for the expression of Prox1 in muscles(35,37). Note that the ocular lymphatics originate from the nasal side, and grow into the limbus (the inner circle) andconjunctiva (outer circles).…”

Fluorescent reporter transgenic mice for in vivo live imaging of angiogenesis and lymphangiogenesis

“…These new mouse models provide opportunities for future research not only in mice but also in other animal models based on genomic conservation between species (e.g., genomic sequences used to create transgenic Prox1-EGFP mice are highly conserved in other species)[73]. For example, Jung et al developed a transgenic rat model for studying surgical physiology along with convenient structural and functional analysis of lymphatic vessels expressing Prox1[43]. …”