Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds (2018)
Figure 6

Fig. 6: Deletion of VE-cadherin leads to selectively increased proliferation of mesenteric LECs.

(A - D) Deletion of VE-cadherin was induced in adult mice (13 weeks) and EdU incorporation into PROX1-positive cell nuclei was determined 6 weeks after Tamoxifen administration. Shown are mesenteric wholemount stainings prepared from control (A, B; n=3) or VE-cadherin-deleted mice (C, D; n=3). (B) and (D) show a magnification of the area outlined by the white dashed line in (A) and (C). Antigens shown in addition to the EdU incorporation are depicted in colour on the left side of the first panel. PEC, PECAM1; PRX1, PROX1. Scale bars = 100 µm.

(E) Enumeration of EdU-positive LECs. The measurements were obtained from three animals and nine confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji. Co-localization of PROX1 and EdU was determined manually in each confocal plane, using the cell counting tool of Fiji. The data represent mean ± SD. **** p ≤ 0.0001. Two-tailed unpaired Student's t test.

(F - I ) Aberrantly enhanced VEGFR-3 phosphorylation in VE-cadherin-deficient mesenteric LECs of six weeks old mice that had received 4-OHT injections at P2 and P4. Shown are wholemount immunostainings for the antigens shown on the left in color. pVR3, pVEGFR-3, PRX1, PROX1; PEC1, PECAM1. (G) and (I) show a magnification of the area outlined by the white dashed box in (F) and (H). Scale bars = 50 μm. The data are representative for 6 (Cdh5lox/lox) and 5 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 2 litters.

(J) Dermal, mesenteric and intestinal tissue pieces of 15 week old control (Cdh5lox/lox) mice were dissociated and total RNA was extracted immediately. For reverse transcription 2.0 µg of total RNA was used for each tissue type. Expression of lymphangiogenic factors VEGF-C and VEGF-D was determined by quantitative real-time (qRT)-PCR using specific TaqMan probes. Samples were measured in triplicate and diagrams are representative for 3 independent experiments. Relative expression (ΔΔCt) of target genes was normalized to the control transcript UBC.

(K, L) Loss of VEGFR-3 surface expression on mesenteric LECs growing in cell sheets was associated with concomitant upregulation of VEGFR-2, scale bars = 50 μm. The data are representative for 4 analyzed animals from 2 litters.VR2, VEGFR-2; VR3, VEGFR-3, PRX1, PROX1.

(M, N) Expression of the transcriptional co-activators YAP and TAZ was analyzed in mesenteric wholemount preparations 8 weeks after induction of VE-Cadherin deletion at 1 year of age by three doses of Tamoxifen. Respective antigens depicted are indicated on the top of the panels in colour. The highly related molecules YAP and TAZ were both recognized by the antibody used and are therefore collectively abbreviated Y/T. Scale bars = 100 μm.

(O) PROX1+ nuclei (red in panels M and N) were classified into YAP/TAZ low, intermediate and high-expressing cells, according to their relative YAP/TAZ immunoreactivity in wildtype and VE-cadherin-deleted mice. The data were obtained from three animals per biological group. PROX1-positive and YAP/TAZ-positive nuclei were counted manually. The pie charts represent mean values.