Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 2. Three-dimensional (3-D) visualization of lymphatic vessels in the portal tract of normal mouse and human livers. A, left: reconstructed 3-D image of the portal tract in the normal mouse liver. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,290 μm 1,290 μm 354 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel), ASMA (white, portal vein), and CK19 (green, bile duct) antibodies. Right: 3-D volume-rendering image of lymphatic vessels and bile ducts using Imaris software with an enlarged image. Scale bars: 100 μm. B: reconstructed 3-D image of the portal tract in normal human liver tissue. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,002 μm 1,168 μm 228 μm. Liver tissue was cleared and stained with PDPN (red, lymphatic vessel) and ASMA (white, portal vein) antibodies. Scale bar: 100 μm. C, left: recon- structed 3-D image of the portal tract in normal human liver tissue. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,193 μm 1,193 μm 233 μm. Liver tissue was cleared and stained with PDPN (red, lymphatic vessel) and CK19 (green, bile duct) antibodies. Right, top: enlarged reconstructed 3-D image. Right, bottom: 3-D volume-rendering image. Scale bars: 100 μm.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Imaging, Staining, Software

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 3. Lymphatic vessels in the capsular area of normal mouse, rat, and human livers. A: comparison of the capsular areas (thickness) among normal human (n ¼ 4), mouse (n ¼ 7), and rat livers (n ¼ 7) stained with Sirius Red. Scale bars: 100 μm. B: two-dimensional (2-D) conventional histology images of the capsular areas (paraffin section, 7-lm thickness) from normal human, mouse, and rat livers stained with PDPN or LYVE1 (red, lymphatic vessel) and Col-I (green, collagenous capsular area), respectively. Yellow arrows indicate lymphatic vessels inside the Col-I positive capsular area. 2-D images were merged with differential interference contrast (DIC) images. Scale bars: 100 μm. C: surface of human liver tissue (top) covered by thin collagenous cap- sule (Glisson’s capsule, yellow arrowhead, bottom) was isolated from normal human liver tissue to visualize capsular lymphatic vessels. D, left: recon- structed 3-D image of lymphatic vessels (PDPN, red) in the capsular area of normal human liver tissue. Imaging condition: z-step size ¼ 0.3 μm, image volume [x] [y] [z] ¼ 1,551 μm 1,551 μm 145 μm. Scale bars: 100 μm. Right: 3-D volume-rendering image. Scale bars: 100 μm.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Comparison, Staining, Isolation, Imaging

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 4. Spatial relationship between lymphatic vessels and bile ducts in biliary cirrhotic livers. A: reconstructed three-dimensional (3-D) image of the portal tract in the liver from a sham-operated mouse (EC-GFP reporter mouse). Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 1,025 μm 1,161 μm 211 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel: LV) and CK19 (white, bile duct: BD). Scale bar: 100 μm. B: reconstructed 3-D image of the portal tract in the liver from a 4-wk BDL mouse (EC-GFP reporter mouse) with a representative single optical sec- tion in the right. Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 862 μm 939 μm 220 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel) and CK19 (white, bile duct). LV, lymphatic vessel; BD, bile duct; PV, portal vein; HA, hepatic artery; PBP: peribili- ary plexus. Scale bars: 100 μm. C: 3-D volume-rendering image of lymphatic vessels and bile ducts in the portal tract with enlarged images (D–F). Scale bar: 100 μm. BDL, bile duct ligation; EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Imaging, Staining, Ligation

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 5. A workflow for quantification of lymphatic vessel parameters in 3-D volume images. Cleared liver tissue was stained with LYVE1 (red) for lym- phatic vessels (LVs) and reconstructed three-dimensional (3-D) images of the portal tract in the mouse liver (EC-GFP reporter mouse) were created. The “Surface” rendering tool in Imaris software were used to determine a total volume of LYVE1-positive LVs (A–C), whereas the “Filament Tracer” rendering tool was used for quantification of the diameter and branch number of LVs (D–F). A:LYVE1-positive LVs were captured based on fluorescence intensities. B: thresholds were manually adjusted to determine LVs. C:Rendered LYVE1-positive LVs were visually checked to confirm proper vascular connections. LVs with improper connections and/or speckled background structure were removed manually. D: LYVE1-positive LVs were captured based on fluores- cence intensities using automatic filament and path detection in the “Filament Tracer” rendering tool. E: thresholds were manually adjusted to determine LVs. F: LVs with disconnected and speckled background structures were removed manually. All outputs of LV parameters were exported to Excel files for statistical analysis. EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Staining, Software

Journal: American journal of physiology. Gastrointestinal and liver physiology

Article Title: An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

doi: 10.1152/ajpgi.00139.2023

Figure Lengend Snippet: Figure 6. Comparison of lymphatic vessel parameters between 3-D volume-rendering images and conventional two-dimensional (2-D) images in normal and biliary fibrotic livers. A, left: reconstructed three-dimensional (3-D) image of the portal tract in the liver from a sham-operated mouse (EC-GFP re- porter mouse). Imaging condition: z-step size ¼ 0.4 μm step, image volume [x] [y] [z] ¼ 777 μm 777 μm 215 μm. Liver tissue was cleared and stained with LYVE1 (red, lymphatic vessel: LV) and aSMA (white, portal vein: PV). Right: 3-D volume-rendering image of lymphatic vessels and portal vein using Imaris software. Scale bars: 100 μm. B, left: reconstructed 3-D image of the portal tract in the liver from a 4-wk BDL mouse (EC-GFP reporter mouse). Imaging condition: z-step size ¼ 0.4 μm, image volume [x] [y] [z] ¼ 793 μm 894 μm 190 μm. Right: 3-D volume-rendering image of lym- phatic vessels and portal vein using Imaris software. Scale bars: 100 μm. C: quantification of lymphatic vessel (LV) parameters (diameter, branch number, and total volume) from 3-D volume-rendering images in sham (n ¼ 4) and 4-wk BDL (n ¼ 3) mouse livers. PT, portal tract; FC, fold change. D: conventional 2-D images of liver tissues (frozen section, 7-lm thickness) from sham and 4-wk BDL mice (EC-GFP reporter mice). Red: LYVE1-positive lymphatic vessel. Scale bars: 100 μm. E: quantification of LV parameters (diameter, number, and total area) from conventional 2-D images in sham (n ¼ 6) and 4-wk BDL (n ¼ 6) mouse livers. BDL, bile duct ligation; EC-GFP, Endothelial-specific green fluorescent protein mice; LV, lymphatic vessel.

Article Snippet: The liver slides were washed with PBS and incubated in blocking buffer (PBS containing 5% donkey serum and 0.3% triton X-100) at room temperature for 1 h. After blocking was completed, the liver slides were incubated with goat anti-mouse LYVE1 (1:200, AF2125, R&D Systems, Minneapolis, MN) (17), sheep anti-rat LYVE1 (1:100, AF7939, R&D Systems) (18), and rabbit anti-COL-I (1:100, ab292, Abcam) (19) primary antibodies at 4 C overnight, followed by washing with PBS for 5 min for 3 times and incubation with secondary antibodies including donkey anti-goat Alexa 647 (1:300, A32849, Invitrogen), donkey anti-sheep Alexa 647 (1:300, A21448, Invitrogen), and donkey anti-rabbit Alexa 488 at room temperature for 30 min. For immunostaining of PDPN in human liver slides, we amplified fluorescence signal using the avidin-biotin amplification method (SP-2001, Vector Laboratories, Newark, CA).

Techniques: Comparison, Imaging, Staining, Software, Ligation

Journal: Genes & Development

Article Title: Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

doi: 10.1101/gad.282400.116

Figure Lengend Snippet: β-Catenin is necessary for the development of LVVs, LVs, VVs, and cardiac valves. ( A – J ) The gene encoding β-catenin ( Ctnnb1 ) was deleted from LECs, LVV-ECs, LV-ECs, and VV-ECs using Lyve1-Cre mice . IHC was performed for the indicated markers. Frontal sections from E14.5 ( A – D ) and E16.5 ( G , H ) embryos revealed the LVVs in controls (arrows) but not in their Lyve1-Cre; Ctnnb1 LOF littermates. Arrowheads point to the valve-forming area of mutants. ( G , H ) At E16.5, VVs are seen in control embryos (yellow arrows) but not in mutants. ( E , F ) SEM confirmed the presence of LVV-ECs (magenta) and VV-ECs (green) in E14.5 control embryos and their absence in Lyve1-Cre;Ctnnb1 LOF littermates. ( I , J ) Whole-mount IHC of the mesenteric lymphatic vessels revealed the presence of LVs in E18.5 control (arrows) but not Lyve1-Cre;Ctnnb1 LOF littermates. ( I ) Furthermore, PROX1 is strongly expressed in the LVs but down-regulated elsewhere in the lymphatic vessels of controls. ( J ) In contrast, PROX1 expression is uniformly high in the lymphatic vessels of the mutants. ( K , L ) Prox1 +/ Cre was used to delete Ctnnb1 from the PROX1 + cells of the cardiac semilunar valves. IHC was performed for the indicated markers. E13.5 Prox1 +/ Cre ; Ctnnb1 LOF embryos lacked the PROX1 + FOXC2 + cells of the cardiac valves (arrows). (LS) Lymph sacs; (IJV) internal jugular vein; (SCV) subclavian vein; (SVC) superior vena cava. Bars: A – J , 200 µm; K , L , 100 µm. n = 4 for each experiment.

Article Snippet: Primary antibodies used for immunostaining were as follows: rabbit anti-PROX1 (AngioBio), goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies (both from Cell Signaling Technologies), goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich).

Techniques: Control, Expressing

Journal: Genes & Development

Article Title: Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

doi: 10.1101/gad.282400.116

Figure Lengend Snippet: β-Catenin is necessary for the patterning of lymphatic vessels. ( A – G ) E17.5 and E14.5 Lyve1-Cre;Ctnnb1 LOF and its control littermates were harvested, and the lymphatic vessels of dorsal skin were analyzed by whole-mount IHC for the indicated markers. ( A , B ) The lymphatic vessels of control embryos have reached and crossed over the midline (red dotted line) to form a network of vessels. In contrast, the lymphatic vessels of the mutants are dilated and have not reached the midline. Additionally, abnormal recruitment of α-SMA + mural cells is visible in the lymphatic vessels of mutants. The lymphatic vessel diameter is quantified in C . ( D , E ) The lymphatic vessels at the leading edge are thin and elongated in E14.5 control embryos. In contrast, they are dilated in Lyve1-Cre;Ctnnb1 LOF embryos. D ′ and E ′ show NRP2 expression alone from the corresponding pictures in D and E , respectively. NRP2 clearly labels the filopodia (red asterisks) on the tip cells of the growing lymphatic vessels. The length ( F ) and the number ( G ) of filapodia are significantly reduced in the mutant embryos. Bars: A , B , 500 µm; D – E ′, 50 µm. n = 4 for each experiment. (**) P < 0.01.

Article Snippet: Primary antibodies used for immunostaining were as follows: rabbit anti-PROX1 (AngioBio), goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies (both from Cell Signaling Technologies), goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich).

Techniques: Control, Expressing, Mutagenesis

Journal: Genes & Development

Article Title: Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

doi: 10.1101/gad.282400.116

Figure Lengend Snippet: The Wnt/β-catenin signaling pathway regulates FOXC2 expression in LECs. ( A – D ) The dorsal skin of E15.5 control ( A , B ) and Lyve1-Cre;Ctnnb1 LOF ( C , D ) embryos was analyzed by whole-mount IHC for the indicated markers. FOXC2 is expressed in both the tip ( A , A ′, arrows) and collecting lymphatic vessels ( B , B ′, arrows) of control embryos. ( B , B ′) Rudimentary LVs are enriched for FOXC2 (arrowhead). ( C , C ′) FOXC2 expression is dramatically down-regulated in the tip cells (arrows) of mutants. ( D , D ′) A modest down-regulation of FOXC2 expression is observed in the collecting lymphatic vessels of mice lacking β-catenin (arrows). Furthermore, LV rudiments are absent in Lyve1-Cre;Ctnnb1 LOF embryos. ( E – G ) The lymphatic vessels of the skin from E16.5 control and Foxc2 −/− littermates were analyzed by IHC using the indicated markers. The magenta lines indicate the distance between the tip cells and the opposing front. ( G ) This distance is significantly increased in Foxc2 −/− embryos, indicating lymphatic vascular hypoplasia. The diameter of lymphatic vessels is also significantly increased in Foxc2 −/− embryos. ( H – J ) The tip cells of E15.5 control embryos have numerous well-formed filopodia (red dots). In contrast, the tip cells of Foxc2 −/− embryos have a bulbous architecture and hardly any filopodia. The number and length of filopodia in control and Foxc2 −/− embryos are quantified in J . Bars: A – D , 100 µm; E , F , 500 µm; H , I , 25 µm. n = 4 for each experiment. (**) P < 0.01; (***) P < 0.001.

Article Snippet: Primary antibodies used for immunostaining were as follows: rabbit anti-PROX1 (AngioBio), goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies (both from Cell Signaling Technologies), goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich).

Techniques: Expressing, Control

Journal: Genes & Development

Article Title: Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

doi: 10.1101/gad.282400.116

Figure Lengend Snippet: FOXC2 compensates for the loss of β-catenin to regulate lymphatic vessel patterning. ( A – E ) Primary human LECs were infected with control or FOXC2-expressing retroviral particles. Scratch assay was performed 24 h later in the presence or absence of 25 µM iCRT3. The space between the red dotted lines indicates the open scratch wound. ( E ) The wound size was measured at various time points and plotted. iCRT3 significantly inhibits the ability of control LECs to “heal” the scratch wound. FOXC2-overexpressing cells are able to significantly overcome iCRT3-induced inhibition. ( F – I ) The lymphatic vessels of the dorsal skin of E16.5 control ( F ), Lyve1-Cre;Ctnnb1 LOF ( G ), Lyve1-Cre;Ctnnb1 LOF ;FOXC2 GOF ( H ), and Lyve1-Cre ;FOXC2 GOF ( I ) embryos were analyzed by IHC for VEGFR3. ( J ) The diameter of the vessels and the distance between the tip cells of the opposing fronts were quantified ( Supplemental Fig. 7E–H ) and plotted. In comparison with control embryos, the lymphatic vessels of Lyve1-Cre;Cttnb1 LOF embryos are significantly dilated. The distance between the migrating fronts is also significantly increased, indicating lymphatic vascular hypoplasia. Ectopic expression of FOXC2 significantly rescues these defects. ( K – N ) Coimmunohistochemistry for the indicated markers revealed the presence of α-SMA + mural cells on the VEGFR3 + lymphatic vessels of Lyve1-Cre;Cttnb1 LOF embryos (shown in L ). ( M ) This defect is fully rescued by ectopic expression of FOXC2. Lower magnification pictures of F – I and K – N are presented in Supplemental Figure 7 . ( O – R ) The mesenteric lymphatic vessels of E17.5 embryos were analyzed by IHC for the indicated markers. Dilation of lymphatic vessels and the abnormal recruitment of α-SMA + mural cells caused by the loss of β-catenin were rescued by FOXC2 overexpression. LVs are seen in control (arrow) but not in any of the mutant embryos. ( S ) Model for the relationship between OSS, β-catenin, FOXC2, and PROX1 during lymphatic vascular development. We did not exclude the role of yet to be identified Wnt ligands that function independently or in cooperation with OSS to activate β-catenin. Bars, 100 µm. ( A – E ) n = 3. ( F – R ) n = 4. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Article Snippet: Primary antibodies used for immunostaining were as follows: rabbit anti-PROX1 (AngioBio), goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies (both from Cell Signaling Technologies), goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich).

Techniques: Infection, Control, Expressing, Retroviral, Wound Healing Assay, Inhibition, Comparison, Over Expression, Mutagenesis