Fig. 5

Vegfr-dependent lymphangiogenic program is required for edema fluid clearance. (A) Schematic diagram illustrating the experimental design for Vegfr inhibition during the osmotic challenge and edema progression. (B) Representative brightfield images showing edema formation in 5 dpf larvae treated with DMSO, 10 µM SAR131675, SAR131675 and 5µM SKLB1002 combined, 2.5 µM CDF in osmotically balanced conditions. (C) Confocal image of craniofacial vessels of a DMSO-treated Tg(lyve1b:dsRed) larva raised in an osmotically balanced condition. (D) Grayscale confocal micrograph of a DMSO-treated larva highlighting the facial lymphatics. (E) Confocal image of trunk vessels of a DMSO-treated Tg(lyve1b:dsRed) larva raised in an osmotically balanced condition. (F) Grayscale confocal micrograph of a DMSO-treated larva highlighting the thoracic duct. (G) Confocal image of craniofacial vessels of a SAR131675-treated Tg(lyve1b:dsRed) larva showing absence of facial lymphatics. (H) Grayscale confocal micrograph of a SAR131675-treated larva highlighting the complete loss of facial lymphatics (I) Confocal image of trunk vessels of a SAR131675-treated Tg(lyve1b:dsRed) larva showing complete absence of thoracic duct. (J) Grayscale confocal micrograph of a SAR131675-treated larva highlighting the loss of thoracic duct. (K) Confocal image of craniofacial vessels of a SAR131675 and SKLB1002 -treated Tg(lyve1b:dsRed) larva showing absence of facial lymphatics. (L) Grayscale confocal micrograph of a SAR131675 and SKLB1002-treated larva highlighting the loss of facial lymphatics (M) Confocal image of trunk vessels of a SAR131675 and SKLB1002 -treated Tg(lyve1b:dsRed) larva showing complete absence of thoracic duct. (N) Grayscale confocal micrograph of a SAR131675 and SKLB1002 -treated larva highlighting the loss of thoracic duct. (O) Confocal image of craniofacial vessels of a CDF-treated Tg(lyve1b:dsRed) larva showing partial absence of facial lymphatics. (P) Grayscale confocal micrograph of CDF-treated larva highlighting the partial loss of facial lymphatics (Q) Confocal image of trunk vessels of CDF-treated Tg(lyve1b:dsRed) larva showing significant disruption of thoracic duct development. (R) Grayscale confocal micrograph of CDF-treated larva highlighting the loss of thoracic duct. (S) Representative brightfield images showing edema outcomes in 6 dpf larvae treated with DMSO, SAR131675, SAR131675 and SKLB1002 combined, or CDF in osmotically balanced or imbalanced conditions. (T) The percentage of edema recovery demonstrated by the ratio of larvae recovered from edema or developed persistent edema at 8 dpf under DMSO and inhibitor treatments in osmotic balance and osmotic imbalance conditions. LFL, Lateral facial lymphatics; MFL, Medial facial lymphatics; LAA, Lymphatic brachial arches; OLV, Otholitic lymphatic vessels. Yellow asterisks indicate missing lymphatic structures in panels F-M. Scale bars = 500 μm (B), 50 μm (C-R).