Arterio-Venous Remodeling in the Zebrafish Trunk Is Controlled by Genetic Programming and Flow-Mediated Fine-Tuning

Ilse Geudens, Baptiste Coxam, Silvanus Alt, Veronique Gebala, Anne-Clemence Vion, Andre Rosa, Holger Gerhardt

Preprint posted on 29 August 2018

Making plans for the future: early zebrafish intersegmental vessels are already pre-patterned to either become bona fide arteries or to be remodelled into veins in a Notch and flow dependent manner.

Selected by Andreas van Impel


Zebrafish is a widely used model system to investigate the molecular control of vascular development. In the zebrafish trunk, intersegmental blood vessels (ISVs) are formed by arterial sprouting and are therefore initially all connected to the Dorsal Aorta (DA). The formation of venous intersegmental vessels is tightly linked to the formation of another endothelial system, the lymphatic vasculature: Sprouting endothelial cells from the Posterior Cardinal Vein (PCV) will either give rise to lymphatic precursor cells or they establish a functional connection to an intersegmental vessel, thereby remodelling it into an intersegmental vein that disconnects from the DA (Fig.1). At the end of this lympho-venous sprouting process, the embryo contains arteries and veins in a robust 50:50 ratio and an additional pool of lymphatic endothelial cells (LECs) that will give rise to the lymphatic vasculature.


Fig.1: Schematic representation of early vascular development in zebrafish. Sprouts from the Posterior Cardinal Vein give rise to both, intersegmental veins and lymphatic precursor cells. (DA: Dorsal Aorta, PCV: Posterior Cardinal Vein, aISV: arterial intersegmental vessel, vISV: venous intersegmental vessel)


How and when the fate of these lympho-venous sprouts is determined to a lymphatic or venous future has been an ongoing area of debate that mainly focused on the role of the transcription factor Prox1 for the specification of a lymphatic cell fate during or even prior to the sprouting from the vein in zebrafish [1-3]. However, to what extent these cell fate decisions might be influenced or even controlled by signals from or within the early intersegmental vessels has not been examined. Furthermore, it is not clear at present by which mechanism the robust overall 50:50 ratio between arteries and veins in the trunk of the developing embryos is achieved, even though the position of arteries and veins along the trunk is not fixed and varies between embryos.


Key findings

In this preprint, Geudens, Coxam and colleagues investigate how the zebrafish embryo achieves the reliable generation of arteries and veins in a 50:50 ratio and whether their specification is controlled by the approaching venous sprouts as has been suggested previously. Live cell imaging of the events during venous sprouting reveals that most sprouts at least transiently form lumenized connections to the pre-existing ISVs, irrespective of whether they will form a venous ISV or whether they will give rise to a lymphatic precursor cell, suggesting that the behaviour of all venous sprouts is the same at this early stage. Since previous results implicated that Notch signalling has an impact on the artery/vein patterning [4], the authors overexpress Notch Intracellular Domain (a constitutive active form of Notch) in ISVs in a mosaic fashion and find that high Notch signalling levels within an early ISV results in the formation of an artery. Importantly, mostly all other wild type ISVs (not expressing the Notch construct) turn into venous ISVs, suggesting that under these conditions a compensation effect kicks in that aims at maintaining the global artery/vein balance. Further experiments show that this compensation is flow dependent as a reduction in blood flow leads to a reduction in the compensation effect. Analysis of the endothelial cell polarity within early ISVs indicates the existence of a differential polarization pattern within future arterial and venous ISVs already prior to the connection with a venous sprout, indicating the existence of a pre-pattern within ISVs that lays down the basis for the future identity of the vessels. In addition, the authors find that the composition of the connection area between early ISVs and the DA is different between individual ISVs: future arteries mostly have a multicellular connection while in future veins the connection with the DA is made by only one cell, a setup that facilitates the regression of this arterial connection once a functional connection to the PCV is established. These and further results show that even the regression of the DA connection is pre-programmed in ISVs and suggest that the ‘decision’, whether or not a secondary sprout will stably connect to an early ISV has already been made before within the ISV (arterial vs. venous fate) and does not depend on a cell fate decision made by the venous sprout (lymphatic vs. venous fate).


Why this is cool

How and when is the cell fate of lympho-venous sprouts in the zebrafish trunk committed to either ‘venous’ or ‘lymphatic’? The emergence and migration of both types of sprouts is triggered by the same pathway, the Vegfc / Vegfr3 signalling axis, and interference with its components leads to a loss of all venous sprouts. In mouse, the transcription factor Prox1 was shown to be the key regulator defining the lymphatic identity of a subset of venous cells within the cardinal vein that subsequently sprout and give rise to the lymphatic lineage. The question whether zebrafish prox1a has a comparable essential role for committing venous cells to a lymphatic cell fate during the migration or even prior to the onset of lympho-venous sprouting has been an ongoing debate in the field.

The Gerhardt lab tackles this important question here from a slightly different angle by investigating whether the ISVs themselves contribute to the cell fate decisions that are made during the formation of definitive arterial or venous ISVs and lymphatic precursor cells. Their central finding is that the early ISVs are not all alike but differ in certain features which indicate that their arterial or venous fate is (to a certain level) already laid down before the sprouts from the PCV emerge. In this model, the pre-pattern within early ISVs consequently dictates the behaviour of the venous sprouts, whether they establish a stable functional connection to the cardinal vein or whether they migrate further and give rise to a lymphatic precursor. Furthermore and in line with another recent preprint [5], the authors identify blood flow and Notch signalling as important regulatory inputs that contribute to the robust generation of a vascular network with a 1:1 ratio of arteries and veins. The presented work therefore provides important new insights on the mechanisms controlling the formation of arteries, veins and lymphatics in zebrafish embryos and on vascular remodelling in general.


Open questions

  • At which point is the pre-pattern within early ISVs generated and what is the molecular mechanism?
  • Is the default fate of a secondary sprout ‘lymphatic’ or how and when is the lymphatic identity of a sprout determined, upon transient connection to a future arterial ISVs?
  • How does the here presented model fit with previous reports that suggested a specification of ‘lymphatic fated’ venous endothelial cells even prior to the onset of venous sprouting? Is there any congruence between the pre-pattern in ISVs and the reported Prox1a pre-pattern within lympho-venous sprouts?


Further reading

  1. Nicenboim, J., Malkinson, G., Lupo, T., Asaf, L., Sela, Y., Mayseless, O., Gibbs-Bar, L., Senderovich, N., Hashimshony, T., Shin, M., Jerafi-Vider, A., Avraham-Davidi, I., Krupalnik, V., Hofi, R., Almog, G., Astin, J.W., Golani, O., Ben-Dor, S., Crosier, P.S., Herzog, W., Lawson, N.D., Hanna, J.H., Yanai, I., Yaniv, K., 2015. Lymphatic vessels arise from specialized angioblasts within a venous niche. Nature 522, 56-61.
  2. Koltowska, K., Lagendijk, A.K., Pichol-Thievend, C., Fischer, J.C., Francois, M., Ober, E.A., Yap, A.S., Hogan, B.M., 2015. Vegfc Regulates Bipotential Precursor Division and Prox1 Expression to Promote Lymphatic Identity in Zebrafish. Cell reports 13, 1828-1841.
  3. van Impel, A., Zhao, Z., Hermkens, D.M., Roukens, M.G., Fischer, J.C., Peterson-Maduro, J., Duckers, H., Ober, E.A., Ingham, P.W., Schulte-Merker, S., 2014. Divergence of zebrafish and mouse lymphatic cell fate specification pathways. Development 141, 1228-1238.
  4. Geudens I, Herpers R, Hermans K, Segura I, Ruiz de Almodovar C, Bussmann J, De Smet F, Vandevelde W, Hogan BM, Siekmann A, Claes F, Moore JC, Pistocchi AS, Loges S, Mazzone M, Mariggi G, Bruyere F, Cotelli F, Kerjaschki D, Noel A, Foidart JM, Gerhardt H, Ny A, Langenberg T, Lawson ND, Duckers HJ, Schulte-Merker S, Carmeliet P, Dewerchin M., 2010. Role of delta-like-4/Notch in the formation and wiring of the lymphatic network in zebrafish. Arterioscler Thromb Vasc Biol. 30(9):1695-1702.
  5. Weijts, B., Gutierrez, E., Saikin, S.K., Ablooglu, A.J., Traver, D., Groisman, A., Tkachenko, E. Blood flow-induced Notch activation and endothelial migration enable embryonic vascular remodeling. bioRxiv preprint first posted online Dec. 19, 2016; doi:

Tags: angiogenesis, cell migration, lymphangiogenesis

Posted on: 24 September 2018 , updated on: 2 October 2018


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