Normal Interstitial Flow is Critical for Developmental Lymphangiogenesis in the Developing Zebrafish

Background: The lymphatic system plays a critical role in the body\u27s fluid and protein homeostasis, immune regulation, and dietary fat absorption. One of the major pathologies of the lymphatic system is primary lymphedema, which occurs in approximately 0.6% of live births and is caused by missing or impaired lymphatic vessels. Although there is a great need for medical intervention into diseases of the lymphatic system, very little is known about its development or how it maintains integrity over time. Recent studies have suggested that biophysical components, such as local extracellular fluid flow, may be important factors during initiation of lymphangiogenesis. We hypothesize that interstitial fluid flow functions as an important morphoregulator during developmental lymphangiogenesis. Methods and Results: In the present study we use pharmacological agents and a mutant fish line to modulate interstitial flow. Our data confirm that a sufficient increase or decrease in interstitial flow can profoundly affect lymphatic patterning and may result in a lymphedema-like phenotype. Proper interstitial flow appears to be necessary during LEC migration for proper lymphatic development. Conclusions: These results support the contention that interstitial flow is an important morphoregulator of developmental lymphangiogenesis

Determination of Lymphatic Vascular Identity and Developmental Timecourse in Zebrafish

Zebrafish lymphatics have been shownto share a number of characteristics with theirhuman counterparts, making the fish a potentiallyuseful model for studying lymphaticdevelopment and disease. The utility of thezebrafish lymphatic model would be substantiallyenhanced by an improved understandingof the spatiotemporal development of theprimary lymphatic vasculature. The goal ofthis project is to identify and map the majorzebrafish lymphatic structures throughoutembryonic to early juvenile stages ofdevelopment. Two transgenic lines, kdrl:RASmCherryxfli1:GFP and stabilin1:YFP,were recently derived to assist in the study ofdeveloping lymphatic vasculature, but theirspecificity has not been rigorously tested.In the course of the present study, weexperimentally validate the utility of these twomarker lines as potential tools for establishinglymphatic vascular identity and visualizingdevelopmental lymphangiogenesis. Weintroduced twenty nanometer red florescentmicrospheres into the blood vasculature offli1:GFP zebrafish and collected tiled opticalz-sections at time intervals spanning earlydevelopment. Three-dimensional reconstructionsof the vasculature were used todifferentiate between blood and lymphaticvessels. Age-matched injected embryos werecompared to the two transgenic lines tofurther assess their specificity. We created aspatiotemporal map of the major lymphaticvessels in the developing zebrafish including apreviously unidentified lymphatic vessel in thegastrointestinal tract. We conclude that thekdr-l:RASmCherryxfli1:GFP line accuratelyidentifies developing lymphatic vessels withthe exception of those associated with thegastrointestinal tract. The stabilin1:YFP line,however, is less reliable, as it marks bothvenous vessels and lymphatic vessels

Determination of Lymphatic Vascular Identity and Developmental Timecourse in Zebrafish

Zebrafish lymphatics have been shownto share a number of characteristics with theirhuman counterparts, making the fish a potentiallyuseful model for studying lymphaticdevelopment and disease. The utility of thezebrafish lymphatic model would be substantiallyenhanced by an improved understandingof the spatiotemporal development of theprimary lymphatic vasculature. The goal ofthis project is to identify and map the majorzebrafish lymphatic structures throughoutembryonic to early juvenile stages ofdevelopment. Two transgenic lines, kdrl:RASmCherryxfli1:GFP and stabilin1:YFP,were recently derived to assist in the study ofdeveloping lymphatic vasculature, but theirspecificity has not been rigorously tested.In the course of the present study, weexperimentally validate the utility of these twomarker lines as potential tools for establishinglymphatic vascular identity and visualizingdevelopmental lymphangiogenesis. Weintroduced twenty nanometer red florescentmicrospheres into the blood vasculature offli1:GFP zebrafish and collected tiled opticalz-sections at time intervals spanning earlydevelopment. Three-dimensional reconstructionsof the vasculature were used todifferentiate between blood and lymphaticvessels. Age-matched injected embryos werecompared to the two transgenic lines tofurther assess their specificity. We created aspatiotemporal map of the major lymphaticvessels in the developing zebrafish including apreviously unidentified lymphatic vessel in thegastrointestinal tract. We conclude that thekdr-l:RASmCherryxfli1:GFP line accuratelyidentifies developing lymphatic vessels withthe exception of those associated with thegastrointestinal tract. The stabilin1:YFP line,however, is less reliable, as it marks bothvenous vessels and lymphatic vessels

Normal Interstitial Flow is Critical for Developmental Lymphangiogenesis in the Developing Zebrafish

Background: The lymphatic system plays a critical role in the body\u27s fluid and protein homeostasis, immune regulation, and dietary fat absorption. One of the major pathologies of the lymphatic system is primary lymphedema, which occurs in approximately 0.6% of live births and is caused by missing or impaired lymphatic vessels. Although there is a great need for medical intervention into diseases of the lymphatic system, very little is known about its development or how it maintains integrity over time. Recent studies have suggested that biophysical components, such as local extracellular fluid flow, may be important factors during initiation of lymphangiogenesis. We hypothesize that interstitial fluid flow functions as an important morphoregulator during developmental lymphangiogenesis. Methods and Results: In the present study we use pharmacological agents and a mutant fish line to modulate interstitial flow. Our data confirm that a sufficient increase or decrease in interstitial flow can profoundly affect lymphatic patterning and may result in a lymphedema-like phenotype. Proper interstitial flow appears to be necessary during LEC migration for proper lymphatic development. Conclusions: These results support the contention that interstitial flow is an important morphoregulator of developmental lymphangiogenesis

Lymphatic regulation in nonmammalian vertebrates

All vertebrate animals share in common the production of lymph through net capillary filtration from their closed circulatory system into their tissues. The balance of forces responsible for net capillary filtration and lymph formation is described by the Starling equation, but additional factors such as vascular and interstitial compliance, which vary markedly among vertebrates, also have a significant impact on rates of lymph formation. Why vertebrates show extreme variability in rates of lymph formation and how nonmammalian vertebrates maintain plasma volume homeostasis is unclear. This gap hampers our understanding of the evolution of the lymphatic system and its interaction with the cardiovascular system. The evolutionary origin of the vertebrate lymphatic system is not clear, but recent advances suggest common developmental factors for lymphangiogenesis in teleost fishes, amphibians, and mammals with some significant changes in the water-land transition. The lymphatic system of anuran amphibians is characterized by large lymphatic sacs and two pairs of lymph hearts that return lymph into the venous circulation but no lymph vessels per se. The lymphatic systems of reptiles and some birds have lymph hearts, and both groups have extensive lymph vessels, but their functional role in both lymph movement and plasma volume homeostasis is almost completely unknown. The purpose of this review is to present an evolutionary perspective in how different vertebrates have solved the common problem of the inevitable formation of lymph from their closed circulatory systems and to point out the many gaps in our knowledge of this evolutionary progression. Copyright © 2013 the American Physiological Society