Ccbe1’s regulation of Vegfc/Vegfr3 signalling during embryonic lymphangiogenesis

1_zebrafish

Merged GFP and DAPI fluorescence image of 6 Sox10-GFP transgenic embryos. Taken by Mariana Herrera Cruz (Instituto de Biotecnología, UNAM, Mexico); the following people also contributed to prepare the sample: Juan Pablo Fernández (INSIBIO (CONICET-UNT), Argentina), Miguel Angel Mendoza (Instituto de Neurobiología, UNAM, Mexico), Paulette Fernández (UNAM, Mexico) and German Sabio (Leloir Institute, Argentina)

Introduction to Vegfr/Vegfr3 in lymphangiogenesis

Lymphangiogenesis is the process where the lymph vasculature develops, and is controlled by Vegfc signal and its receptor Vegfr3. The balance of the Vegfc/Vegfr3 signalling pathway is crucial to the lymphatic development as serious phenotypes can develop as a result of inappropriate expression.

For Vegfc, overexpression in the skin of mice has led to hyperplasia of lymphatic vessels whereas Vegfc knockout mice develop lymphedema due to accumulation of fluid. In humans, mutations in the VEGFC and VEGFR3 genes have been linked to lymphedema as well (Gordon et al., 2013).

Lymphangiogenesis in Zebrafish

Lymphangiogenesis begins when precursor cells from the cardinal vein form the parachordal lymphangioblasts (PLs) which then travel, both dorsally and ventrally, along the arteries, remodelling into the major trunk lymphatic vessels, thoracic duct (TD) and other important vessels. Vegfr3 and Vegfc is essential for venous angiogenesis, like the migration of the precursor cells and also the development of the PLs.

Discovery of Ccbe1 and its role in lymphangiogenesis

Ccbe1 (collagen and calcium-binding EGF domains 1) was found in zebrafish mutants which were missing lymphatic but not blood vascular development. It was shown that ccbe1 acts at the same stage of development as vegfc and vegfr3 (Hogan et al., 2009a). Ccbe1 deficiency in mice do not have lymphatic vasculature while CCBE1 mutations in humans see similar phenotypes like VEGFC/VEGFR3, where lymphedema and also lymphangiectasia develops. Past experiments have detected links between the interaction of both Ccbe1 and Vegfc, but details of their mechanism have yet to be elucidated.

Overview of Ccbe1 activation of Vegfc

Ccbe1 activates Vegfc to induce Vegfr3 signaling. Brief overview of the interaction between Ccbe1, Vegfc and Vegfr3.

Genetic interaction between Ccbe1, Vegfc and Vegfr3

  • Homozygous mutants see complete lack of vasculature
Fig2A-H

Fig 2A-H. Mutants seen here are all homozygous. Arrows show the thoracic duct formation, while * indicates a loss of structures when compared with the wildtype.

  • Heterozygotes from previous observations indicate that wild-type lymph development is seen with partial loss of ccbe1 or vegfr3.
Fig2I-L

Fig 2I-L. Percentage of genotype (WT, single/double/triple heterozygotes) against TD length.

  • Fig 2I indicates that for TD length ≤50%, 71% of embryos were double heterozygotes for vegfc/vegfr3 (2J: 54% ccbe1/vegfr3, 2K: 61% ccbe1/vegfc), implying that there is interaction between the genes to rescue the phenotype from homozygous mutants.
  • Gene interaction here shows that lymph development depends on the level of Vegfc/Vegfr3 activation as well as on Ccbe1.

ccbe1 mutation suppresses phenotypes induced by exogenous expression of Vegfc/Vegfr3 signalling in arteries and veins

Fig3A-C

Fig 3A-C. (A) At 72 hpf, dll4morphants display an arterial hyperbranching phenotype (arrow) driven by increased Vegfc/Vegfr3 signaling in the transgenic Tg(fli1a:EGFP)line. This phenotype was suppressed in ccbe1 mutants. (B) In dll4 morphants, arteries are sensitized to increased vegfc expression during primary sprouting. Arteries in MO-dll4, Vegfc mRNA-injected embryos display aberrant, ectopic turning (arrow) as early as 30 hpf. Embryos from ccbe1 carrier incrosses, injected with vegfc mRNA and MO-dll4, were sorted into the phenotypic categories ‘wild type’ and ‘severe’. (C) Confocal projections of Tg(fli1a:EGFP; flt1:tomato; hsp70l:Gal;4XUAS:vegfc)embryos show that endothelial cells in heat-shocked embryos display aberrant ectopic branching at 72 hpf. The ectopic endothelial cells are venous derived (arrow in Ciii). Heat-shocked embryos that were injected with MO-ccbe1 do not show this phenotype (asterisks).

Artery Experiments

  • Dll4 gene knockdown leads to excessive intersegmental artery angiogenesis (aISV) and aISV hyperbranching  by 72 hours as Dll4 reduces Vegfc/Vegfr3 signalling by inhibition of the pathway .
  • ccbe1 heterozygous embryos with knocked-down dll4 were used to examine the effects of ccbe1 on aISV.
  • Population with greatly reduced or no hyperbranching were significantly ccbe1 mutants (Fig 3Aiii and graph: Fig 81%, p<0.0001) while those with severe hyperbranching were mainly wild-type and heterozygotes (Fig 3Aii and graph, 83%, p=0.0019)
  • Exogenous Vegfc was added via mRNA injection with dll4 morpholinos into wildtype embryos which saw turning of aISVs in the trunk
  • ccbe1 heterozygous mutants with similar treatments displayed weak phenotypes for the turning of aISVs (Fig3Biii, 70%, p<0.0001)
  • Embryos displaying severe phenotypes was mainly wild-type and heterozygous embryos (Fig 3B and graph, 83%, p=0.0006)
  • These results indicate that ccbe1 mutations suppressed the dll4 loss-of-function phenotype

Venous Experiments

  • A new transgenic zebrafish line was generated with full-length vegfc expression being controled by hsp701 promoter
  • Following heat shocks at 28 and 56 hpf (hours post fertilization), veins emerge ectopically, resulting in hyperbranched intersegmental vessels (vISVs)
  • ccbe1 morpholino injection rescued the vISV phenotype, blocking excessive venous development (Fig 3Civ and graph, P<0.0001)

 

 

Ccbe1 is required for Vegfr3-dependent Erk activation in embryonic veins

Fig4A-C

Fig 4A-C. (A) Expression of phospho-Erk (P-Erk) at 32 hpf. P-Erk is indicated by green area and signal increases in Vegfc-induced embryos. Cross section merged channel images shown in a and b, P-Erk only in c and d. Treatment with the Erk inhibitor PD98059 led to a reduction in all P-Erk staining. Arrows indicate P-Erk expression in the dorsal posterior cardinal vein (PCV). DA (dorsal aorta) and PCV (posterior cardinal vein) are indicated.
(B) Comparison of P-Erk staining in control uninjected (left), MO-vegfr3 and MO-ccbe1 embryos. Upper panels are merged images and lower P-Erk only, viewed laterally (left) and cross-sectioned (right). Cross sections (right) are from separate embryos. Arrows indicate P-Erk expression in the dorsal PCV. DA and PCV are indicated. (C) Quantification of P-Erk-positive cells in the cardinal vein located in the dorsal compared with ventral wall (left-hand graph). Scores through individual sections of z-stack images from 12 control embryos, scored laterally across three somites in the trunk. Quantification of P-Erk-positive cells in the cardinal vein in control and MO-injected conditions (right-hand graph) (scores from n=10 control embryos, n=13 MO-vegfr3-injected and n=15 MO-ccbe1-injected embryos)

 

  • Vegfr3 is known to signal via activation of intracellular kinases like Erk
  • Immunofluorescence was used to examine phosphorylation activity of Erk. A specific signal was created via adding an inhibitor of Erk phosphorylation, allowing only Vegfr3 activation.
  • MO-vegfr3 and MO-ccbe1 were injected into embryos and compared with wild-type to see their effect on the phenotype.
  • Both morpholino injections yielded a reduced in Erk activation as see in Fig 4B and 4C

Ccbe1 enhances Vegfc-driven sprouting

Fig5A

Fig 5A. Image of ccbe1 and vegfc overexpression in the floor plate, leading to ectopic turning of ISVs. Arrows indicate accumulation of endothelial cells at dorsal aspects of the embryo.

  • two other transgenic lines were generated, which expressed ccbe1 or vegfc from the shh promoter in the floor plate (FP), leading to overexpression of either protein.
  • Individual lines showed no phenotype but when the lines were crossed, aberrant ectopic turning of ISVs was seen at 32 hpf.
  • At 48 hpf, ccbe1 lines still saw no phenotype while vegfc lines saw endothelial cell accumulation at the horizontal myoseptum and ectopic sprouting of ISVs (Fig 5A)
  • Double mutants at 48 hpf saw more severe phenotypes, indicating that ccbe1 expression further enhances vegfc phenotypes.

Conclusion

All in all, the experiments performed shows that ccbe1 genetically interacts with vegfc and vegfr3 and mutation or deletions in ccbe1 suppresses the formation of aISV or vISV when Vegfc/Vegfr3 signalling is disrupted. It can be concluded that Ccbe1 has a important role to play in terms of regulating Vegfc/Vegfr3 signalling when it comes to lymphangiogenesis.

 

References

  1. Le Guen, L., Karpanen, T., Schulte, D., Harris, N.C., Koltowska, K., Roukens, G., Bower, N.I., van Impel, A., Stacker, S.A., Achen, M.G., Schulte-Merker, S., Hogan, B.M.. Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis. Development 2014 141:1239-1249; posted ahead of print February 12, 2014,doi:10.1242/dev.100495
  2. Gordon, K., Schulte, D., Brice, G., Simpson, M. A., Roukens, M. G., van Impel, A., Connell, F., Kalidas, K., Jeffery, S., Mortimer, P. S. et al.(2013). Mutation in vascular endothelial growth factor-C, a ligand for vascular endothelial growth factor receptor-3, is associated with autosomal dominant milroy-like primary lymphedema. Circ. Res.112, 956-960
  3. Hogan, B. M., Bos, F. L., Bussmann, J., Witte, M., Chi, N. C., Duckers, H. J. and Schulte-Merker, S.(2009a). Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting. Nat. Genet. 41, 396-398

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