Figure 3. Hdac3 deficiency causes impaired lymphatic drainage and anomalous lymphatic valve development in mesenteric lymphatic vessels.

(A and B) Evans blue dye injection into the left hind paw (A and B, white arrows) of P5 Hdac3^Cdh5KO mice showed reflux (retrograde flow) into the tail, right hind limb, and abdomen (A and B, red arrows) compared with an absence of reflux in the control mice (black arrows). Original magnification ×2.5. (C and D) P5 Hdac3^Cdh5KO mice showed Evans blue dye reflux (red arrows) into dermal lymph vessels and mesenteric lymph nodes compared with control mice (dotted line, black arrows). Original magnification ×3. (E) Control P5 mice showed normal unidirectional cephalad drainage restricted to the thoracic duct (black arrow). Hdac3^Cdh5KO mice exhibited reflux into the intercostal lymphatics lateral to the thoracic duct in the thoracoepigastric region (red arrows) and reduced drainage into the thoracic duct (green arrow). (F) Coimmunofluorescence staining for Lyve1 (green) and smooth muscle actin (SMA) (red) showed abnormal smooth muscle recruitment (white arrows) to blood-filled dermal lymphatic capillaries in P6 Hdac3^Cdh5KO mice compared with controls. (G) Quantitation of lymphatic valve territories in E17.5 Hdac3^Lyve1KO mesenteric lymphatic vessels. The P value was determined by unpaired Student’s t test. (H–J) Whole-mount coimmunofluorescence staining of Hdac3^Lyve1KO (H and I) and Hdac3^Cdh5KO (J) mesenteric lymphatic vessels showed a lack of Prox1-expressing (red) lymphatic valves in Pecam1⁺ (green) lymphatic vessels (yellow arrows) compared with controls (white arrows). (K and L) Whole-mount coimmunofluorescence staining of Hdac3^Lyve1KO (K) and Hdac3^Cdh5KO (L) mesenteric lymphatic vessels showed a lack of Itga9-expressing (green) lymphatic valves in Prox1⁺ (red) lymphatic vessels (yellow arrows) compared with controls (white arrows). (M and N) Whole-mount coimmunofluorescence staining of Hdac3^Lyve1KO (M) and Hdac3^Cdh5KO (N) mesenteric lymphatic vessels showed excessive SMA (red) coverage (yellow arrows) compared with controls (white arrows). Vegfr3 (green) was used as a lymphatic vessel marker. Data represent the mean ± SEM and are representative of 3 independent experiments. Sub-stacks of Z-stack images are presented in H–N. Scale bars: 100 μm (F) and 1 μm (H–N). See also Supplemental Figure 6.