De novo blood vessel formation occurs through coalescence of endothelial cells (ECs) into a cord-like structure, followed by lumenization either through cell-1,2,3 or cord-hollowing4,5,6,7. Vessels generated in this manner are restricted in diameter to one or two ECs, and these models fail to explain how vasculogenesis can form large-diameter vessels. Here, we describe a model for large vessel formation that does not require a cord-like structure or a hollowing step. In this model, ECs coalesce into a network of struts in the future lumen of the vessel, a process dependent upon bone morphogenetic protein signalling. The vessel wall forms around this network and consists initially of only a few patches of ECs. To withstand external forces and to maintain the shape of the vessel, strut formation traps erythrocytes into compartments to form a rigid structure. Struts gradually prune and ECs from struts migrate into and become part of the vessel wall. Experimental severing of struts resulted in vessel collapse, disturbed blood flow and remodelling defects, demonstrating that struts enable the patency of large vessels during their formation.
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Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
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We thank the Animal Facility for zebrafish care (UCSD and Hubrecht Institute). We thank D. Yelon (University of California, San Diego) for the silent heart morpholino, S.-W. Jin (Yale Cardiovascular Research Center) for generously providing us with the hsp70l:noggin plasmid, J. Bakkers (Hubrecht Institute) for the hsp70:bmp2b line, J. den Hertog for the bre:egfp line and P. Tsai for use of the Q-bio laboratory confocal system. We thank J. Santini, N. Gohad (Zeiss) and K. Fertig (Leica) for microscopy technical assistance, the UCSD School of Medicine Microscopy Core, the Princess Máxima Imaging Center and the Hubrecht Institute Optical Imaging Center. This work was supported by the San Diego School of Medicine Microscopy Core (P30 NS047101). We thank R. van der Linden (Hubrecht Institute) for help with cell sorting. Part of this work was supported by a European Research Council grant (ERC project no. 220-H75001EU/HSCOrigin-309361; C.R.), a TOP subsidy from NWO/ZonMw (912.15.017; C.R.), NIH/NINDS R01NS108472 (I.S. and D.K.), NIH/NINDS R35NS097265 (I.S. and D.K.) and NIH R01DK074482 (D.T.).
The authors declare no competing interests.
Peer review information Nature Cell Biology thanks M. Luisa Iruela-Arispe and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
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a, All ECs are marked by both laCherry (magenta; cytoskeleton) and eGFP (green; nuclei). Struts are depicted by arrowheads. Images are representative of 20 embryos analysed from two independent experiments. b, Quantification of the number of endothelial struts at 22 hpf and 26 hpf by confocal microscopy and represented as the median values with the first and third quartiles; the whiskers represent the minimum and maximum (4 different clutches per time point (n = 26; n = 25; n = 27; n = 25 for 22 hpf and n = 25; n = 28; n = 25; n = 25 for 26 hpf. Two-tailed Student’s t-test. ***P < 0.0001 for all 4 tests). Arrowheads depict endothelial struts. Images are representative of indicated groups from four independent experiments. c, Strut formation in the anterior part of the PCV at 22hpf by confocal microscopy and represented as the median values with the first and third quartiles; the whiskers represent the minimum and maximum (n = 30 embryos from three independent experiments). Arrowheads depict endothelial struts. d, Quantification of the number of acridine orange positive ECs in the CV region at 20 hpf and representative confocal images. White arrowhead depicts a positive EC (n = 20 embryos from two independent experiments). Scale bars are defined in the figure.
a, Rescue of ephrinb2a morphants with efnb2a mRNA. Embryos were grouped by the level of rescue (near full, partial or no rescue) of the floor of the DA, indicated by the asterisks. (n = 71 embryos analysed in one experiment, n numbers per group indicated in graph). Images are representative of 5 embryos analysed by confocal microscopy per group. b, Representative images and quantification of the number of struts in embryos treated with the Notch inhibitor DAPT or injected with an ephnb2a MO. As a control, embryos were injected with a scrambled MO. (n = 14 embryos per condition). Data presented as the median values with the first and third quartiles; the whiskers represent the minimum and maximum. Images are representative of 5 embryos analysed by confocal microscopy per condition. Scale bars are defined in the figure.
a, Mosaic expression of laClover under the control of the EC specific promoter fli1a. Arrowhead indicates mClover positive strut at 22 hpf (upper panel). Lower panel shows an image of the same region at 48 hpf. Images are representative of 9 embryos analysed from three independent experiments. b, Gating strategy used to FACS sort ECs from dissected CV regions followed by qPCR (10 CVs were pooled per condition measured in two independent experiments). Of note, the bmp pathway is active in multiple tissues at this time, including erythrocytes and strong activity in epithelial cells (Fig. 3e asterisks), these cells are included in the unsorted CV (black bars). Scale bars are defined in the figure.
a, Gating strategy used to FACS sort ECs from dissected CV regions followed by qPCR (b) The onset of blood flow was inhibited by either injecting of a cardiac troponin T2a (tnnt2a) MO or through administration of 3x concentrated ms-222 (tricaine methanesulfonate). Erythrocytes are marked by GFP (globin) and all ECs by laCherry (magenta). Data are presented as the median values with the first and third quartiles; the whiskers represent the minimum and maximum. One-way analysis of variance (ANOVA) with Dunn’s post-hoc test. ns = not significant (n = 25 embryos per time points per experiment of three independent experiments). Images are representative of 5 embryos analysed by confocal microscopy per condition per experiment. c, 48 hpf embryo injected with tnnt2a MO. Asterisks indicate an incomplete formation of the CV wall. Images are representative of 10 embryos analysed from two independent experiments. Scale bars are defined in the figure.
a, Laser ablation of a single strut resulted in a slight deformation of the CV, shown by the superimposed image of all timepoints. Stills are representative of 18 embryos analysed from three independent experiments. Scale bar is defined in the figure (b) Schematic. 1) Venous (blue) and arterial (red) EC precursors originate from distinct locations within the lateral plate mesoderm, with the primitive erythrocytes (precursors) positioned medially adjacent to the venous ECs. 2) ECs and erythrocytes migrate towards the midline of the embryo. Arterial ECs migrate along and directly contact the ventral face of the somites, thereby they receive inductive cues that strengthens the arterial fate (shades of red). 3) At the midline, venous and arterial ECs coalesce into a network of struts and form a common precursor vessel. This process is bmp2b dependent, which is secreted by, among others, the erythrocytes. Strut formation encloses erythrocytes into compartments. 4) The vessel wall consists initially only out of a few patches of ECs and upon pruning of struts, ECs from struts migrate into and are incorporated in the vessel wall. 5) Arterial ECs participating in strut formation have a weak arterial identity, which progressively increases (shades of red) and results in the expression of the Notch target efnb2b, which drives the unmixing of arterial and venous ECs. Segregation of these arterial and venous ECs results in the formation of the DA and CV 6) Onset of circulation flushes the bmp2b expressing erythrocytes from the CV, which is an important step for complete pruning of all struts. The lumen of the CV is now maintained by blood pressure rather than through the support of struts.
Supplementary Table 1, list of primers used. Supplementary Table 2, list of reagents used.
Formation of the caudal vein.
The dorsal aorta and caudal vein are formed from a common precursor vessel.
Ectopic expression of bmp2b prevents pruning of struts.
Pruning of endothelial struts results in the gradual release of erythrocytes into circulation.
Laser ablation of endothelial struts.
Visualization of the caudal vein post ablation.
Endothelial struts maintain the shape of the caudal vein lumen.
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Weijts, B., Shaked, I., Ginsberg, M. et al. Endothelial struts enable the generation of large lumenized blood vessels de novo. Nat Cell Biol 23, 322–329 (2021). https://doi.org/10.1038/s41556-021-00664-3
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