DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport

Abstract

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.

Figure 10. DLL4 promotes continuous lacteal regeneration in the adult small intestine.

(A) During normal homeostasis the intestinal villi are subject to many stressors, including dietary fat, the gut microbiota, and villus movements, which lacteals encounter as chylomicrons, TLR ligands, and mechanical stress, respectively. In normal mice, VEGFC/D signaling, through VEGFR2 and VEGFR3, promotes lacteal DLL4 expression and Notch signaling, thus maintaining LEC survival, sprouting, and migration to replace damaged cells. Furthermore, LECs in lacteals have both continuous and discontinuous adherens junctions, balancing the need for continuous regeneration with the need for efficient chylomicron uptake. (B) In the absence of DLL4, lacteals are unable to undergo remodeling and have impaired survival and migration and thus are not able to maintain the normal length in villi. Lymphatic Dll4 deletion also promotes a shift from mixed adherens junctions to mostly continuous junctions, contributing to inefficient chylomicron uptake and transport. MLN, mesenteric lymph node.

Figure 9. Lymphatic DLL4 is necessary for fat uptake and discontinuous lacteal adherens junctions.

(A) Quantification of plasma TGs and FFAs in olive oil–gavaged WT and Dll4^ΔLEC mice. Plasma TG and FFA (mean ± SD) at indicated time points in WT and Dll4^ΔLEC mice; n = 3. (B) Representative whole-mount images of submucosal lymphatic capillaries (LYVE1, green; VE-cadherin, red) from control and Dll4^ΔLEC mice. Arrows indicate button-like junctions that are present in both WT and Dll4^ΔLEC submucosal lymphatic capillaries. (C) Top: Representative whole-mount images of lacteals (LYVE1, green; VE-cadherin, red) from control and Dll4^ΔLEC mice. White arrowheads, continuous junctions; white arrows, discontinuous junctions. Bottom: Representative high-magnification images of single lacteal LECs (VE-cadherin, black) from control and Dll4^ΔLEC mice. (D) Quantification of lacteals with continuous, discontinuous, or mixed staining for VE-cadherin from control and Dll4^ΔLEC mice. Percentage (mean ± SD) of lacteals in each group from control and Dll4^ΔLEC mice; n = 5. Scale bars: 10 μm, B and C (top); 5 μm, C (bottom). *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t test.

Figure 8. DLL4 expression is dependent on active VEGFR3 and VEGFR2 signaling.

(A) Whole-mount immunostaining of DLL4 (white, arrow) on lacteals (LYVE1, red) of mice treated with control IgG, αVEGFR2, or αVEGFR3 blocking antibodies. Perinuclear DLL4 intensity/lacteal volume (mean ± SD); n = 3. (B) Serum-starved LECs transfected with control, VEGFR2, VEGFR3, and VEGFR2/VEGFR3 (R2+R3) siRNA were treated for 24 hours with either BSA or VEGFC and analyzed by Western blotting. Data are representative of 2 independent experiments. (C) Serum-starved LECs were treated with BSA, VEGFC, and VEGFC C156S for 24 hours and analyzed by Western blotting. Data are representative of 2 independent experiments. (D) Quantification of lacteal filopodia after αVEGFR2 or αVEGFR3 treatment. LEC filopodia/villus (mean ± SD); n = 3. (E) Representative whole-mount images of intestinal villus blood capillaries (PECAM1, green) and lacteals (LYVE1, red) after αVEGFR2 or αVEGFR3 treatment. Lacteal length (mean ± SD) binned for given lengths after 2 weeks of antibody administration; n = 3. Scale bars: 10 μm, A; 50 μm, E. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed unpaired Student’s t test (E) or 1-way ANOVA with Bonferroni correction (A and D).

Figure 7. DLL4 is necessary for efficient LEC migration.

(A) Individual LEC migration was tracked for 20 hours after a wound was made in a LEC monolayer transfected with either control or DLL4-specific siRNA and treated with BSA or VEGFC. Tracks in red or black represent LECs that traveled less than or more than the average actual distance (avg. dist.), respectively. Data shown are representative of 3 independent experiments. (B) Total LEC track distance (mean ± SD) in arbitrary units (a.u.); n = 3. (C) Actual LEC distance (mean ± SD) traveled in arbitrary units; n = 3. (D) Directionality (actual/total distance; mean ± SD); n = 3. *P < 0.05, **P < 0.01, 2-tailed unpaired Student’s t test.

Figure 6. DLL4 is required for LEC lacteal tip cell position and survival.

(A) DLL4 expression is important for tip cell competence. Control (Prox1-CreERT2 Rosa26-EYFP) and Dll4^ΔLEC (Dll4^fl/fl Prox1-CreERT2 Rosa26-EYFP) mice were injected with a reduced amount of tamoxifen to induce mosaic Cre activation. Representative images from WT and Dll4^ΔLEC mosaic lacteals (YFP, green; PROX1, red; LYVE1, blue.) (B) Decreased number of Dll4-deficient LECs. Percentage (mean ± SD) of YFP⁺ LECs in mosaic Cre recombination control and Dll4^ΔLEC Rosa26-YFP mice; n = 4. (C) Quantification of recombined YFP⁺ control or Dll4^ΔLEC cells in tip position. Percentage (mean ± SD) of YFP⁺ lacteal tip cells in mosaic Cre recombination control and Dll4^ΔLEC Rosa26-YFP mice; n = 4. (D) Quantification of recombined YFP⁺ control or Dll4^ΔLEC cells position on vertical axis of the lacteal. Position of YFP⁺ lacteal cells in mosaic Cre recombination control and Dll4^ΔLEC Rosa26-YFP mice as a percentage (mean ± SD) of lacteal length (lacteal base = 0, lacteal tip cell = 100); n = 4. (E) DLL4 depletion in vitro promotes apoptosis. Quantification of caspase-3⁺ LECs (caspase-3, red; β-catenin, green; DAPI, blue) 48 hours after transfection with two different siRNAs targeting DLL4. Mean percentage of caspase-3⁺ LECs. The graph represents pooled data of 2 independent experiments. Scale bars: 25 μm, A; 20 μm, E. *P < 0.05, 2-tailed unpaired Student’s t test.

Figure 5. DLL4 is required for lacteal length maintenance.

(A) Loss of DLL4 protein in Dll4^ΔLEC lacteals. Whole-mount immunofluorescence staining for DLL4 (red) and VEGFR3 (green) in WT and Dll4^ΔLEC lacteals. (B) Efficient loss of Dll4 mRNA in Dll4^ΔLEC LECs. Dll4 expression (mean ± SD) in sorted intestinal LECs from WT and Dll4^ΔLEC mice analyzed by RT-qPCR; n = 3. (C) Dll4 inactivation reduces lacteal filopodia (LYVE1, red). LYVE1 is intentionally overexposed to highlight lacteal filopodia (white dots). Filopodia/lacteal (mean ± SD) of control and Dll4^ΔLEC mice 10 weeks after tamoxifen injection; n = 4–5. (D) Lacteals are shorter in Dll4^ΔLEC mice. Representative images of villus blood capillaries (PECAM1, green) and lacteals (LYVE1, red) from control and Dll4^ΔLEC mice after 10 weeks of Dll4 deletion. Lacteal length (mean ± SD) binned for given lengths 3 weeks (top) and 10 weeks (bottom) after tamoxifen injection; n = 3. Scale bars: 10 μm, A; 20 μm, C; 100 μm, D. *P < 0.05, ***P < 0.001, 2-tailed unpaired Student’s t test.

Figure 4. DLL4 expression and Notch signaling are high in adult lacteals.

(A) DLL4 (white) displays perinuclear (arrows) and surface localization on villus arterioles. Endomucin (green) marks villus venules. Endothelial cells were identified by staining for zonula occludens 1 (ZO1, red). (B) DLL4 (white) is highly expressed on tip cells of villus blood capillaries (PECAM1, green). (C) DLL4 (white) is highly expressed on tip cells of villus lacteals (LYVE1, red); DAPI, blue. Dotted staining outside of lacteals is likely due to shedding from intestinal epithelial cells, where DLL4 was previously reported to be highly expressed (82). Relative DLL4 intensity (mean ± SD) in lacteal tip cells compared with neighboring stalk cells; n = 3. (D) Villus arterioles (VEGFR2, blue, arrowheads) have higher levels of Notch reporter (Venus, green) compared with venules (MAdCAM-1, red, arrows) in CBF1:H2B-Venus mice. Percentage (mean) Venus intensity of MAdCAM-1–negative arterioles versus MAdCAM-1–positive venules; n = 2. (E) Whole-mount immunostaining of lacteals (LYVE1, blue) and Venus (green) from adult CBF1:H2B-Venus mice. All LEC nuclei (PROX1, red) were also Venus⁺. Scale bars: 50 μm, A and D; 10 μm, B and C; 20 μm, E. **P < 0.01, 2-tailed unpaired Student’s t test.

Figure 3. Lacteal regeneration in the adult small intestine.

(A) Adult lacteals, but not dermal lymphatic capillaries (LYVE1, red), display filopodia. Filopodia/lymphatic capillary (mean ± SD) in adult intestine and ear skin; n = 3–4. S.I., small intestine. (B) A small proportion of lacteal LECs are Ki67⁺ (PROX1⁺Ki67⁺DAPI⁺, white nuclei, arrowhead), while submucosal LECs (PROX1⁺DAPI⁺, pink nuclei, arrow) are quiescent. Percentage (mean ± SD) of Ki67⁺ LECs in the submucosal and lacteal lymphatic vessels of the small intestine; n = 8. (C) Adult ear skin LECs are quiescent (PROX1⁺DAPI⁺, pink nuclei). Percentage (mean ± SD) of Ki67⁺ LECs in adult ear skin; n = 4. (D) Embryonic skin LECs are actively proliferating (PROX1⁺Ki67⁺, yellow nuclei, arrowheads). Quiescent LECs are Ki67^– (PROX1⁺DAPI⁺, pink nuclei, arrows). Percentage (mean ± SD) of Ki67⁺ LECs in E16.5–E17.5 embryonic skin; n = 3. (E) Relative lacteal length is constant along the entire small intestine. Whole-mount immunostaining of lacteals (LYVE1, red) and intestinal blood capillaries (PECAM1, green) from different intestinal segments of adult C57BL/6 mice. (F) The number of lacteals per villus (mean ± SD; n = 4) decreases from the duodenum to the ileum. (G) Quantification of relative lacteal length in the indicated parts of small intestine. Lacteal length/blood capillary cage length (mean ± SD); n = 4. (H) Quantification of lacteal filopodia in different parts of intestine. Filopodia/lacteal (mean ± SD); n = 4. Scale bars: 20 μm, A–D; 100 μm, E. *P < 0.05, 2-tailed unpaired Student’s t test with Welch’s correction. duo, duodenum; jej, jejunum.

Figure 2. Immune cells and specialized ECM organization in adult small intestinal villi.

Confocal microscope images of adult mouse small intestinal villi after whole-mount immunostaining. (A) DCs (CD11c, red) are closely associated with SMC fibers (αSMA, cyan). Inset: ×5 3D Surpass projection of DCs (CD11c, red) interacting with SMC fibers (arrowheads). DCs associated with SMC fibers can be observed sampling the intestinal lumen (arrow). Macrophages are shown in green (F4/80). (B) Fibronectin (green) is highly expressed on villus SMCs (αSMA, red). (C) Expression of periostin (red) and tenascin C (green) is restricted to the crypts (*) and villi, respectively. Scale bars: 50 μm, A–C.

Figure 1. Unique organization of small intestinal stroma.

Confocal microscope images of adult mouse small intestinal villi after whole-mount immunostaining. (A) A dense blood capillary network (VEGFR2, green) lies in close proximity to intestinal epithelial cells (E-cadherin, red). (B) Adult lacteals (LYVE1, red) and villus blood vessels (PECAM1, green) in adult small intestine. (C) Pericytes (NG2, green, arrowheads) are closely associated with villus blood capillaries (VEGFR2, red). (D) αSMA (red) staining reveals a network of SMCs in the villi, located inside the blood capillary network (green). MM, muscularis mucosa; CM, circular muscle; LM, longitudinal muscle. (E) A subset of villus SMCs (αSMA, cyan) are in close proximity to lacteals (LYVE1, red). (F) Higher-magnification view of near lacteal villus SMCs (αSMA, cyan). (G) Terminal ends of villus SMCs (αSMA, red) interact with villus blood vessels (PECAM1, green). Nuclei are stained with DAPI (blue). Ep, epithelial cells. Scale bars: 50 μm, A–D; 20 μm, E; 10 μm, F; 5 μm, G.