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Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment of venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.
Fig. 3.
xNR2F2 knockdown impairs LEC commitment, migration and function (A and B) Stage (ST)45 tadpoles injected with non-silencing (‘ns’) (A) or Xenopus (x)NR2F2 morpholino (Mo; B) showing edema (arrowheads) in the latter. (C and D) WISH on ST32 tadpoles injected with ns (C) or xNR2F2 Mo (D), revealing less prox1 signal in area 1 (a1), indicating reduced LEC commitment [12]. (E–G) WISH for prox1 on ST35/36 tadpoles treated with ns (E, white in G) or xNR2F2ATG Mo (F, black in G) and corresponding quantification (G), revealing significantly lower prox1 signal in area 1 (a1; indicating LEC commitment), a2 and a3 (indicating LEC migration) and a significantly reduced maximal migration distance (MMD) in xNR2F2 morphants (G; data represent mean area or distance ± SEM; N = 20; ∗P < 0.05, ∗∗P < 0.01 vs. ns Mo). (H and I) Lymphangiograms (arrows indicate injection spot) of ST46 Tg(Flk1:eGFP) tadpoles revealing draining defects in the VCLV of xNR2F2 Mo-injected tadpoles (I). Bottom panels are higher magnifications of the boxed areas. Tadpolehead facing left and dorsal side facing up in C-F and H and I. Scale bars: 200 μm in E and F; 250 m in C,D,H and I and 1 mm in A and B.
Figure S1. Nr2f2 is expressed in vECs and LECs in zebrafish and Xenopus
A-C. Whole-mount in situ hybridization (WISH) on 48 hpf (A,B) or 6 dpf (C) zebrafish (z)
showing znr2f2 expression in the posterior cardinal vein (PCV: arrowheads in B and lower
luminal structure in inset (âiâ)1), the parachordal lymphangioblast (PL) string (a bilateral
structure at notochord (NC) level; arrowheads in i2) and the thoracic duct (TD; arrowhead in
i3) in addition to its expression in the neural tube (NT). Note lack of expression in the dorsal
aorta (DA). D. WISH on stage (ST)40 Xenopus tadpoles revealing xnr2f2 expression in the
PCV and the ventral caudal lymphatic vessel (VCLV), but not in the DA. E. After specifically
labeling LECs with red fluorescent TRITC-dextran (shown in the inset where VCLV ECs
appear yellow) in ST45 Tg(Flk1:eGFP) tadpoles, TRITC+GFP+ LECs (enriched for LEC
markers prox1 and flt4; yellow bars) and TRITC-GFP+ blood vascular ECs (BECs; green
bars) were sorted and assayed by qRT-PCR. Data expressed as % relative to the
expression in the GFP- non-EC fraction (simultaneously sorted with the other fractions)
represent the mean ± SEM of 7-10 independent sorting experiments (*P<0.05; **P<0.01 vs.
BECs). Scale bars: 25 μm in A,C,D; 100 μm in B and 200 μm in E (inset).
Figure S3. Scoring of lymphatic phenotype upon xNR2F2 knockdown
A. The top panel represents a schematic of the main vascular structures in stage (ST)45
Xenopus tadpoles (blood vessels are in blue, dorsal aorta or DA is highlighted in red). For
evaluation of the lymphatic vessels in Xenopus Tg(Flk1:eGFP) tadpoles, the eGFP+ main
lymphatic vessels in the trunk region (the dorsal and ventral caudal lymphatic vessel or
DCLV and VCLV, respectively, shown in green) were evaluated. Since the severity of the
phenotype was different along the anterior-posterior axis, anterior and posteriortrunk
regions were scored separately for the (partial) absence or abnormalities of DCLV or VCLV.
The lower insets show the quantitative analysis of the phenotype in the anterior region (left)
or posterior region (right) revealing a dose-dependent effect of Xenopus (x)NR2F2
morpholino (Mo) as compared to the non-silencing (‘ns’) Mo- treated tadpoles on the
presence/disorganized structure of the DCLV (left diagram) and the VCLV (right diagram).
Numbers on top of the bars indicate the numbers of tadpoles analyzed (*P<0.0001 vs. ns Mo
for each of the xNR2F2 Mo doses). B-E. Representative confocal images of the anterior
portion of the trunk of ST45 Tg(Flk1:eGFP) tadpoles injected with ns (B,C = inset) or
xNR2F2 Mo (D,E = inset) showing absence (asterisks in D) of large parts of the dorsal
caudal lymphatic vessel (DCLV) or disorganized structure (E) of the ventral caudal lymphatic
vessel (VCLV). Tadpolehead facing left and dorsal side facing up in B-E. Scale bars: 200
μm in C,E; 250 μm in B,D.
Figure S5. Endothelial cell sorting from Xenopus tadpoles
A. Fluorescent image of the trunk region of a stage (ST)45 Tg(Flk1:eGFP) Xenopus embryo
(both lymphatic and blood vessels are GFP+) injected 1 day earlier with red TRITC-dextran.
The bottom picture zooms in on the posterior cardinal vein (PCV) and the ventral caudal
lymphatic vessel (VCLV) revealing specific TRITC labeling of the lymphatic endothelial cells
(LECs) of the VCLV (in yellow). B. The FACS dot plot shows the sorting set-up for isolating
TRITC+eGFP+ LECs and TRITC-eGFP+ blood vascular endothelial cells (BECs). C. Diagram
represents qRT-PCR analysis on sorted BECs, LECs and non-ECs (eGFP-) fractions
revealing significant enrichment for EC marker flk1 in both BEC (green bars) and LEC
(yellow bars) fractions compared to the non-EC fraction (red bars) and enrichment for LEC
markers (prox1 and flt4) in the LEC fraction vs. the BEC and non-EC fractions. Data
expressed as % vs. non-ECs represent the mean ± SEM of 7-10 independent sort
experiments. (*P<0.05, **P<0.01 vs. non-ECs or BECs). Scale bars: 200 μm in A.
