An Evolutionarily Conserved Role for Polydom/Svep1 During Lymphatic Vessel Formation

Abstract

Rationale: Lymphatic vessel formation and function constitutes a physiologically and pathophysiologically important process, but its genetic control is not well understood.

Objective: Here, we identify the secreted Polydom/Svep1 protein as essential for the formation of the lymphatic vasculature. We analyzed mutants in mice and zebrafish to gain insight into the role of Polydom/Svep1 in the lymphangiogenic process.

Methods and results: Phenotypic analysis of zebrafish polydom/svep1 mutants showed a decrease in venous and lymphovenous sprouting, which leads to an increased number of intersegmental arteries. A reduced number of primordial lymphatic cells populated the horizontal myoseptum region but failed to migrate dorsally or ventrally, resulting in severe reduction of the lymphatic trunk vasculature. Corresponding mutants in the mouse Polydom/Svep1 gene showed normal egression of Prox-1⁺ cells from the cardinal vein at E10.5, but at E12.5, the tight association between the cardinal vein and lymphatic endothelial cells at the first lymphovenous contact site was abnormal. Furthermore, mesenteric lymphatic structures at E18.5 failed to undergo remodeling events in mutants and lacked lymphatic valves. In both fish and mouse embryos, the expression of the gene suggests a nonendothelial and noncell autonomous mechanism.

Conclusions: Our data identify zebrafish and mouse Polydom/Svep1 as essential extracellular factors for lymphangiogenesis. Expression of the respective genes by mesenchymal cells in intimate proximity with venous and lymphatic endothelial cells is required for sprouting and migratory events in zebrafish and for remodeling events of the lymphatic intraluminal valves in mouse embryos.

Keywords: Polydom/Svep1; arteries; lymphangiogenesis; lymphatic vessels; mice; veins; zebrafish.

Figure 1.

Characterization of a zebrafish mutant that affects the formation of the lymphatic vascular system. A, Gross morphology of the Ly02-512 mutant. The mutant embryos appear without major macroscopic defects or developmental delay, and form a swim bladder at 5 d post-fertilization (dpf). However, closer examination shows abnormalities in the jaw (arrowhead), and the embryo displays edema around the heart, eye, and intestine (arrows). B, Analysis of the vasculature in a fli1a:GFP; flt1^enh:RFP transgenic background, highlighting arteries (red). Note the normal overall patterning of the blood vasculature despite the higher proportion of intersegmental arteries in the mutant embryo. C, Mutant embryos lack all or most aspects of the thoracic duct (TD), which in wild-type siblings is positioned just ventral to the dorsal aorta (DA; white arrows in C and D) but is absent in mutants (asterisks). D, Rhodamine dextran injection into the cardinal vein shows normal blood circulation in Ly02-512 mutants.

Figure 2.

Ly02-512 encodes an allele of polydom/svep1. A, Positional cloning approach to identify the molecular lesion in Ly02-512 mutant embryos. Using polymorphic markers, Ly02-512 was mapped to a region between markers z4.29 and z.7.4 comprising ≈350 kb on linkage group 7. B, Three different BAC constructs that were independently inserted as transgenes into the Ly02-512 line containing 12 candidate genes within the linkage group. C, In Ly02-512 mutant embryos which contained the DKEY-8E16 BAC, the TD phenotype was rescued (P<0.001), suggesting DKEY-8E16 to contain the gene of interest. D, Two other mutant lines identified in the forward genetic screen, Ly04-093 and Ly05-265, failed to complement Ly02-512 and each other, suggesting that they represent alleles of the same gene. E, Sequencing of the 3 independent mutant alleles revealed 3 different nonsense substitutions, predicted to result in truncated versions of the Polydom/Svep1 protein. F, Schematic presentation of the Polydom/Svep1 protein domain structure and the positions of the truncations in the 3 mutant lines. Red rectangle: signal peptide; blue pentagon: von Willebrand factor type A domain; yellow ovals: SUSHI repeat; green pentagons: epidermal growth factor (EGF)–like and calcium-binding EGF-like domains; and pink hexagon: pentraxin domain.

Figure 3.

Polydom/svep1 mutants show reduced venous and lymphovenous sprouting. A and F, Quantification of sprouts from the posterior cardinal vein (PCV) in wild-type (wt) siblings and polydom/svep1 mutants, in plcg1 morphant embryos. Knockdown of plcg1 suppresses arterial formation, hence only venous structures can be observed in a fli1a:GFP transgenic background. Heterozygous embryos show a significant reduction in venous sprouting events from the PCV, and this is further exacerbated in mutant embryos at 54 h post-fertilization (hpf; wt siblings: n=29, heterozygous embryos: n=53, and polydom/svep1 mutants: n=26). B, Still frames from confocal time-lapse imaging of a wt sibling and polydom/svep1 mutant embryo in a fli1a:GFP; flt1^enh:RFP double transgenic background are shown over the course of 32.5 to 46.5 hpf. Both the number of secondary sprouts from the PCV (yellow arrowheads) and parachordal lymphangioblast (PL) cells (white arrowheads) were reduced in mutant embryos. C and G, polydom/svep1 mutant embryos form a reduced number of PLs at the horizontal myoseptum (HMS) region. Confocal images of wt sibling and polydom/svep1 mutant embryos at 48 hpf in fli1a:GFP;flt1^enh:RFP background and quantification of PLs at 54 hpf (wt siblings: n=8, heterozygous embryos; and n=11, polydom/svep1 mutants: n=6). D, PL cells at the level of the HMS fail to migrate along intersegmental arteries in the polydom/svep1 mutants. Still frames from confocal time-lapse imaging of a wt sibling (a–c) and a polydom/svep1 mutant embryo (d–f) in a fli1a:GFP transgenic background are shown over the course of 2.5 to 3.5 d post-fertilization (dpf). E and H, An increased number of arterial intersegmental vessels (ISVs) at the expense of venous ISVs in polydom/svep1 mutants is highlighted by flt1^enh:RFP expression in fli1a:GFP background at 5 dpf (siblings: n=20, polydom/svep1 mutants: n=10). Values are presented as means±SD. **P<0.01; ***P<0.001.

Figure 4.

Posterior cardinal vein (PCV) cells express pERK and parachordal lymphangioblast (PL) cells express Prox-1 in svep1 mutant embryos. A, Partial maximal projection of antibody staining against Prox-1 (red) and fli1a:GFP (green) in embryos from an svep1^+/−; fli1a:GFP incross at 48 h post-fertilization (hpf). Prox-1–positive PL cells are indicated by an arrowhead. B, Quantification of Prox-1–positive PL cells across 9 somites at the horizontal myoseptum (HMS) in siblings (96 out of 106 counted PLs are Prox-1 positive in 18 embryos) and svep1 mutants (28 out of 30 PLs are Prox-1 positive in 8 mutant embryos). C–E, pERK-positive cells were quantified in the PCV by scoring RFP and GFP coexpression (indicated by arrows) laterally across 6 somites in the trunk. C, In ccbe1 morpholino (MO) injected embryos (total number of 6 pERK-positive cells in 5 ccbe1 morphants; total number of 36 pERK-positive cells in 6 uninjected controls) and (D) vegf-c^hu6410 mutants (total number of 17 pERK-positive cells in 7 mutants and 67 pERK-positive cells in 8 siblings), the amount of pERK-positive cells is significantly reduced, whereas in svep1 mutants (E and F) no difference in pERK can be detected in the PCV (total number of 151 pERK-positive cells in 31 siblings compared with 48 pERK-positive cells in 9 svep1 mutants). F, Partial maximal projections of antibody staining against pERK (red) and fli1a:EGFP (green) in svep1^+/−; fli1a:GFP incrosses show no difference in the amount of pERK-positive cells in the PCV at 32 hpf. Bar graphs show mean±SD. For statistical analysis, the Mann–Whitney test was applied in all panels.

Figure 5.

Zebrafish polydom/svep1 is expressed dynamically at regions of venous and lymphatic endothelial cell migratory activity. A, The first polydom/svep1 expression appears around 34 h post-fertilization (hpf) in nonendothelial cells along the dorsal aorta (DA) and posterior cardinal vein (PCV), as depicted by svep1:Gal4FF;UAS:GFP expression in a kdrl:mCherry transgenic background. B, By 48 hpf, the number of polydom/svep1-positive cells along the PCV has increased, and polydom/svep1-positive cells can abundantly be found in the immediate vicinity of both arterial (highlighted in red in flt1^enh:RFP background) and venous (marked with asterisks) intersegmental vessels (ISVs). C, Higher magnification of an independent region at 48 hpf, demonstrating the tight connection between endothelial cells (red) and polydom/svep1-positive cells (green). D–F, Between 48 and 72 hpf, when parachordal lymphangioblast (PL) cells populate the horizontal myoseptum (HMS) region and start to migrate dorsally and ventrally along arterial ISVs, individual cells in the midline of the embryo start to express polydom/svep1. The polydom/svep1-expressing cells are in a immediate contact with migrating PL cells. G, Cross-section of a 72 hpf svep1:Gal4FF;UAS:GFP embryo in a kdrl:mCherry background. Note the close association of PL cells (red) and polydom/svep1-positive cells (green) in the HMS region (white arrow heads), and of PCV cells and polydom/svep1-positive cells (blue arrow head). The dorsal aorta (red arrow head) is not covered by polydom/svep1-positive cells at this time point any more. H, Still frames of a confocal time-lapse imaging of a svep1:Gal4FF;UAS:GFP;kdrl:mCherry embryo from 36 to 51 hpf. Polydom/svep1-positive cells (green) and PL cells (red) start appearing at the horizontal midline region around 39 hpf. Note the almost simultaneous appearance of both cell types. The full movie can be seen online. I, At 3 and 5 d post-fertilization (dpf), polydom/svep1 expression can be seen in between the DA and the PCV, exactly in the region, which the lymphatic cells of the future thoracic duct (TD) will populate.

Figure 6.

Characterization of Polydom/Svep1 function in mice. A, Targeting construct to create a Polydom/Svep1 knockin mouse. The LacZ construct disrupts the locus and is predicted to result in a protein truncated after amino acid 559. The primers used for genotyping are indicated as fw, mt-rv, and wt-rv. B, Genotyping using the PCR primers clearly distinguishes the wild-type from the mutant allele. C, E18.5 litters from heterozygous parents contained mutant and sibling embryos in normal Mendelian ratios. Heterozygous siblings are indistinguishable from wild-type embryos, but mutant embryos show a clear nuchal edema (arrow). FRT indicates flippase recognition target sites; fw, forward primer; loxP (locus of X-over P1), recognition sites for Cre recombinase; mt-rv, mutant reverse primer; and wt-rv, wild-type reverse primer.

Figure 7.

The first detectable abnormalities in Polydom/Svep1 mutant embryos occur at the level of lymphovenous valve formation. To determine the first phenotypic abnormalities in mutant embryos, we used whole-mount imaging on the ultramicroscope at E11.0, E12.5, and E13.5 embryos. Combined antibody staining highlighting CD31, Prox-1, and Vegfr-3 expression at E11.0 (A and B), and Prox-1 and Vegfr-3 at E12.5 (C–H) did not reveal any alterations between wild-type sibling and mutant embryos. In all cases examined, we could not detect differences in future lymphatic endothelial cells (LECs) egressing from the cardinal vein (CV), and the formation of the primitive primordial thoracic duct (pTD) and the peripheral longitudinal lymphatic vessel (pLLV) seemed unchanged. However, we did notice at E12.5 a difference at the level of the first lymphovenous connection: here, Prox-1–positive cells of the CV and Prox-1–positive LECs come together in tight association (G), but this association was not found in mutant embryos (H). I and J, Closer view of the lymphovenous contact site in volume reconstructions at E13.5. Whereas in wild-type embryos the dual contact sites are massive structures composed of multiple cells, in Polydom/Svep1 deficient embryos only few individual cells are actually in contact to the high Prox-1–positive expression domain inside the CV. Scale bars correspond to 100 μm in all panels.

Figure 8.

Polydom/Svep1 mutant embryos fail to remodel mesenteric lymphatic vessels at E18.5 and do not form valves. Lymphatic structures in the mesenteries of mutant embryos are closely associated with blood vessels as they are in siblings, but they are significantly smaller in size, appear nonluminized and do not contain valve structures. A–J, Whole-mount immunofluorescent staining of mesenteric vessels for Vegfr-3, CD31, and Prox-1 at E18.5 of a Svep1^+/− and Svep1^−/− embryo. At this time of the development, the maturation of lymphatic vessels is well underway, including the formation of lymphatic valve structures that retain high levels of Prox-1 protein. CD31 and VEGFR-3 levels are downregulated in the lymphatic vessels but remain high in the lymphatic valve regions. A, Overlay picture of VEGFR-3, CD31, and Prox-1 staining with the boxed region shown in higher magnification in B–E; F–J, Immunofluorescent staining of vessels in a E18.5 Svep1^−/− embryo indicates that the size of the lymphatic vessel is dramatically decreased. Prox-1 expression remains high in the lymphatic endothelial cells (LECs), and lymphatic valves fail to form. F, Overlay picture of VEGFR-3, CD31, and Prox-1 staining with the boxed area shown in higher magnification in G–J. Scale bars correspond to 50 μm in all panels.