Determination of lymphatic vascular identity and developmental timecourse in zebrafish (Danio rerio)

Zebrafish lymphatics have been shown to share a number of characteristics with their human counterparts, making the fish a potentially useful model for studying lymphatic development and disease. The utility of the zebrafish lymphatic model would be substantially enhanced by an improved understanding of the spatiotemporal development of the primary lymphatic vasculature. The goal of this project is to identify and map the major zebrafish lymphatic structures throughout embryonic to early juvenile stages of development. Two transgenic lines, kdr-1:RASmCherryxfli1:GFP and stabilin1:YFP, were recently derived to assist in the study of developing lymphatic vasculature, but their specificity has not been rigorously tested. In the course of the present study, we experimentally validate the utility of these two marker lines as potential tools for establishing lymphatic vascular identity and visualizing developmental lymphangiogenesis. We introduced twenty nanometer red florescent microspheres into the blood vasculature of flil:GFP zebrafish and collected tiled optical z-sections at time intervals spanning early development. Three-dimensional reconstructions of the vasculature were used to differentiate between blood and lymphatic vessels. Age-matched injected embryos were compared to the two transgenic lines to further assess their specificity. We created a spatiotemporal map of the major lymphatic vessels in the developing zebrafish including a previously unidentified lymphatic vessel in the gastrointestinal tract. We conclude that the kdr-1:RASmCherryxfli1:GFP line accurately identifies developing lymphatic vessels with the exception of those associated with the gastrointestinal tract. The stabilin1:YFP line, however, is less reliable, as it marks both venous vessels and lymphatic vessels.