title: Inactivation of FAK plus one allele of Cdc42 resulted in lymphatic vessel and blood vessel misconnection.
title: FAK, Cdc42
titleType: AlternativeTitle
Creators: name: Peng, Xu
Publisher: name: Zenodo
Year: 2024

description: Legends:

Figure 1. Cdc42 was required for lymphatic development during embryogenesis. Gross examination of P1 pups (A, B) and E18.5 embryos (C, D). P1 Cdc42Cdh5KO (KO) pups showed severe edema with purple skin (B), but P1 Cdc42flox/flox control (CO) pups presented no signs of edema with pink skin (A). Side view of E18.5 KO embryos showed edema, but no edema was displayed in CO embryos (C). Histological analysis (H&E) on the dorsal skin of E18.5 CO (E) and KO embryos (F). The subcutaneous tissue of E18.5 KO embryos exhibited distinct expansion (red line) (F). Immunofluorescence staining with VEGFR3 (G, H), Lyve1 (I, J), and CD31 (K, L) on CO (G, I, K, M) and KO embryos (H, J, L, N). The dilated lymphatic vessels are indicated by white arrows. M and N are merged images. Bars: 125 μm (F), 100 μm (N).

 

Figure 2. Loss of Cdc42 prevented LEC filopodia formation and increased the lumen size of lymphatic vessels in the skin. Double immunostaining of E17.5 Cdc42flox/flox control (CO) (A-D) and Cdc42Cdh5KO (KO) embryonic skin (E-H) with CD31 (B, F) and VEGFR3 (A, E). C and G are merged images. D and H are higher magnification views of the boxed regions that show the filopodia (yellow arrows) in the CO (D), but not in the KO (H). (I) Quantification analysis of lymphatic lumen size displays the significant increase in the KO embryos (O). Double immunostaining of E18.5 CO (I-K) and KO embryonic skin (L-N) with CD31 (J, M) and Lyve1 (I, L). K and N are merged images. The lymphatic lumen size was increased in KO (G, N), compared to the CO (C, K). Bars: 100 μm.

 

Figure 3. Cdc42 was essential for collecting lymphatic vessel maturation and lymphatic valve formation. Double immunofluorescence staining of Lyve1 (A, D) and CD31 (B, E) on the mesentery of E18.5 Cdc42flox/flox control (CO) (A, B, C) and Cdc42Cdh5KO (KO) (D, E, F). The lymphatic lumen size of KO (F) was larger than that of CO (C). Lyve1 (G, J) and smooth muscle actin (H, K) double staining on P1 CO (G, H, I) and KO mesentery (J, K, L). Smooth muscle actin positive cells were recruited in P1 CO lymphatic (I), but not in KO (L). Double immunofluorescence staining of Prox1 (M, P) and CD31 (N, Q) on E18.5 CO (M, N, O) and KO mesentery (P, Q, R). Prox1 expression was increased in the lymphatic valve formation region but expressed at a comparable level in the whole lymphatic (M, O). P1 mesenteric vessels were stained with Prox1 (S, V) and CD31 (T, W). U and X are merged images. P1 lymphatic valves formed mature leaflets with high Prox1 expression in the CO (U), but no lymphatic valves were evident in the KO (X). Bar: 100 μm.

 

Figure 4. Inactivation of Cdc42 increased VEGFR3 levels in the mesenteric collecting lymphatic vessels. Whole-mount immunofluorescence staining with antibodies against VEGFR3 (A, D, G, J) and CD31 (B, E, H, K) on E17.5 (A-F) and P1 (G-L) mesenteries. VEGFR3 levels slightly increased at E17.5 (D) and dramatically increased at E18.5 (J) in Cdc42Cdh5KO (KO), compared to Cdc42flox/flox control (CO) (A, G). Bar: 100 μm.

 

Figure 5. Inactivation of Cdc42 disrupted cell-cell adhesion in vivo and in vitro. Whole-mount immunofluorescence staining with antibodies against Prox1 (A, D) and VE-Cadherin (A-F) on E17.5 control (A-C) and Cdc42Cdh5KO (D-F) mesenteries. Images with higher magnification showed more condense aggregates in Cdc42Cdh5KO embryos (F, Yellow arrows) while VE-Cadherin was more evenly distributed in control animals (C). (G-V) Confocal images of cultured HDLECs stained with ZO-1 (G, I, K, M), VE-cadherin (H, I, L, M) and DAPI (I, J, M, N). J and N are the higher magnification views in I and M respectively. siCdc42-treated HDLECs display discontinuous cell-cell junctions (M, N), whereas the junctions in siControl appear continuous and thicker (I, J). (O-V) Confocal images of cultured HDLECs stained with Phalloidin (O, Q, S, U), ZO-1 (P, Q, T, U) and DAPI (Q, R, U, V). R and V are higher magnification shown in Q and U. Consistently, siCdc42-treated HDLECs had disrupted cell-cell boundaries, and discontinuous ZO-1-positive junctions colocalized with aberrant organization of Phalloidin-positive F-actin (U, V). Bars: 20𝛍m.

 

Figure 6. Deletion of Cdc42 in lymphatic endothelial cells resulted in embryonic edema with dilated lymphatic vessels in the skin. The Prox1-CreERT2;Cdc42flox/flox pregnant mice were injected with Tamoxifen (5 mg) at E10.5 and E11.5. Gross examination of E15.5 Cdc42flox/flox control (CO) (A) and Cdc42Prox1KO (KO) (E) embryos. KO embryos displayed edema in the dorsal skin. Double immunostaining of E15.5 CO (B-D) and KO (F-H) embryonic skin with VEGFR3 (B, F) and CD31 (C, G). D and H are merged images. Bars: 100 𝛍m.

 

Figure 7. Knocking down Cdc42 in HDLECs impacted VEGFc-induced FAK phosphorylation. (A) HDLECs cell lysates were harvested after VEGFc treatment. PBD pull down assay and Western blots were then performed. The active Cdc42 was detected by Cdc42 antibodies and Vinculin was blotted as a loading control. (B) HDLECs were transfected with scrambled siRNA or Cdc42 siRNA and stimulated with VEGFc. Cell lysates were incubated with Human Phospho-Kinase Array membranes. a1-2, a17-18, g1-2 reference spots. b5, b6 FAK spots.

 

Figure 8. Inactivation of FAK with hypomorphic Cdc42 resulted in profound embryonic edema with dilated lymphatic vessels. Gross examination of E18.5 FAKflox/wt; Cdc42flox/+; Cdh5-Cre+ (A), FAKflox/flox; Cdc42flox/+ control (E) and FAK knockout plus Cdc42 hypomorphis (FAKflox/flox; Cdc42flox/+; Cdh5-Cre+) embryos (I). Whole-mount staining on E18.5 FAKflox/wt; Cdc42flox/+; Cdh5-Cre+ (B, C, D), FAKflox/flox; Cdc42flox/+ control (F, G, H) and FAK knockout plus Cdc42 hypomorphs (J, K, L) embryonic skin with Lyve1 (B, F, J) and CD31 (C, G, K) antibodies. Bars: 100 𝛍m.

 

Figure 9. Inactivation of FAK with hypomorphic Cdc42 in ECs induced blood-filled lymphatic vessels. Gross examination of whole pups (A, E) and dorsal skin (B, F) at P1 of control (FAKflox/flox; Cdc42flox/+) (A, B) and EC FAK knockout with hypomorphic Cdc42 (FAKflox/flox; Cdc42flox/+; Cdh5-Cre+) (E, F)). Black arrows indicate blood-filled lymphatic vessels. (C, D, G H). Immunofluorescence double staining of Lyve1 (C, G) and CD31 (D, H) on the skin of E18.5 control (C, D) and FAK knockout with hypomorphic Cdc42 (G, H). White arrows indicate spherical lymphatic vessels that present only in the embryos with blood-filled lymphatic vessels. 

Bars: 100 𝛍m.

 

Figure S1. Deletion of Cdc42 in ECs. Sections from E12.5 Cdc42flox/flox control (CO) (A, B) and Cdc42Cdh5KO (KO) embryos (C, D) were stained with CD31 (A, C) and Cdc42 (B, D). Cdh5-Cre+: RosamT/mG E12.5 embryo were stained with GFP (E, H) and CD31 (F, I). E18.5 mesentery was stained with GFP (K) and CD31 (L). Panel G, J and M are merged pictures. Yellow arrows point to GFP negative vascular ECs. Bars: 100 𝛍m.

 

Figure S2. Cdc42 is required for blood vessel formation. Gross examination of Cdc42flox/flox control (CO) (A, B, E, F, I, J) and Cdc42Cdh5KO (KO) (C, D, G, H, K, L) embryos at E12.5 (A-D), E13.5 (E-H) and E14.5 (I-L) with or without york sac.  

 

Figure S3. Inactivation of Cdc42 impaired blood vessel remodeling in the brain. Whole-mount staining with CD105 was performed on E11.5 Cdc42flox/flox control (CO) (A, C) and Cdc42Cdh5KO (KO) embryos (B, D). Red arrows indicate abruptly disrupted blood vessels. Bar: 50 μm.

 

Figure S4. Inactivation of Cdc42 impaired neonatal mice lung inflation. H&E staining anaylsis was performed on P1 Cdc42flox/flox control (CO) (A, C) and Cdc42Cdh5KO (KO) pups (B, D). (C and D) Higher magnification microphotographs of CO (C) and KO (D). CD31 and Lyve1 costaining on E17.5 embryo thoracic sections (E, F). The yellow arrows point to the dilated lympahtic vessels. Bars: 500 𝛍m (A and B) and 250 𝛍m (C and D), 100 𝛍m (E and F). 

 

Figure S5. Deletion of Cdc42 resulted in lymphatic vessel dilation. Whole-mount immunofluoresence staining was performed on E14.5 Cdc42flox/flox control (CO) (A, B, C) and Cdc42Cdh5KO (KO) (D, E, F) embryonic skin with CD31 (A, D) and VEGFR3 (B, E). Bars: 100 𝛍m.

 

Figure S6. Inactivation of Cdc42 impaired lymphatic valve formaiton. Whole-mount immunofluoresence staining was performed on E16.5 (A-F) and E17.5 (G-L) mesentery with Prox1 (A, D, G, J) and CD31 (B, E, H, K) antibodies. Bars: 100 𝛍m.

 

Figure S7. Deletion of Cdc42 prevented lymphatic valve formation. Whole-mount immunofluoresence staining was performed on P1 Cdc42flox/flox control (CO) (A, B, C) and Cdc42Cdh5KO (KO) (D, E, F) mesentery with CD31 (A, D) and integrin α9 (B, E) antibodies. Bars: 100 𝛍m.

 

Figure S8.  Knockdown of Cdc42 in HDLECs impaired VE-Cadherin distribution. (A-H) Confocal images of cultured HDLECs stained with ZO-1 (Green), VE-cadherin (Red) and DAPI (Blue). D, H are the higher magnification view of boxed region shown in C and G respectively. siCdc42 -treated HDLECs display discontinuous cell-cell junctions (H, yellow arrows), whereas the junctions in siControl appear continuous and thicker. Bars: 20 𝛍m.

 

Figure S9. Knockout FAK in the vascular endothelial cells did not cause edema during embryo development. Gross examination of E12.5 embryos (A, B) and E18.5 embryos (C, D). E12.5 (B) and E18.5 FAK knockout embryos (D) did not show edema, similar as their littermate control (A, C)