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Endothelial cell self-fusion during vascular pruning.

Lenard A, Daetwyler S, Betz C, Ellertsdottir E, Belting HG, Huisken J, Affolter M - PLoS Biol. (2015)

Bottom Line: Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail.In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection.Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum der Universität Basel, Basel, Switzerland.

ABSTRACT
During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.

No MeSH data available.


Related in: MedlinePlus

SIV development and maturation.(A–D) A model representing four phases of SIV plexus development in the zebrafish embryo between ~36 and 84 hpf. The SIV plexus is blue, and the dorsal aorta (DA), posterior cardinal vein (PCV), and intersegmental vessels are marked in grey. Single endothelial cells sprout ventrally and separate from the PCV (A) to form a primary SIV branch (B). Angiogenic sprouts grow out of the primary SIV and fuse to each other, forming a reticular plexus with multiple cross branches (C, red) and vascular loops (C, green); simultaneously, the plexus grows and moves ventrally. Eventually, the cross branches (and hence the loops) are removed, and the plexus simplifies, forming a set of parallel vertical branches draining into a large ventral SIV branch (D). (A’–E’) Stills from a SPIM time-lapse movie representing five phases of SIV development corresponding to models in A. (A”–E”) Stills from a SPIM time-lapse movie representing SIV development in a silent heart morphant embryo corresponding to models in A. In this case, the SIV keeps its reticular structure because of impaired pruning. (F) A graph comparing the SIV vascular loop formation and remodeling in a wild-type (grey) and silent heart embryo (orange), based on SPIM time-lapse experiments between 36 and 84 hpf. From the left, showing the number of cross branches pruned during remodeling phase, the number of cross branches/loops closed via collateral fusion, the number of cross branches/loops remaining until the end of the movie, and the sum of all loops observed throughout the movie. The values are average numbers per SIV plexus with standard deviation (n = 19 for wild type [WT] and n = 9 for silent heart [SIH]). *** p < 0.001. (G) A graph showing the percentage contribution of pruned (grey), closed by collateral fusion (orange), and remaining (blue) vascular loops to all events observed in WT versus silent heart embryos. See also S1 and S2 Figs, S1–S4 Movies, S1 Table and S1 Data.
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pbio.1002126.g001: SIV development and maturation.(A–D) A model representing four phases of SIV plexus development in the zebrafish embryo between ~36 and 84 hpf. The SIV plexus is blue, and the dorsal aorta (DA), posterior cardinal vein (PCV), and intersegmental vessels are marked in grey. Single endothelial cells sprout ventrally and separate from the PCV (A) to form a primary SIV branch (B). Angiogenic sprouts grow out of the primary SIV and fuse to each other, forming a reticular plexus with multiple cross branches (C, red) and vascular loops (C, green); simultaneously, the plexus grows and moves ventrally. Eventually, the cross branches (and hence the loops) are removed, and the plexus simplifies, forming a set of parallel vertical branches draining into a large ventral SIV branch (D). (A’–E’) Stills from a SPIM time-lapse movie representing five phases of SIV development corresponding to models in A. (A”–E”) Stills from a SPIM time-lapse movie representing SIV development in a silent heart morphant embryo corresponding to models in A. In this case, the SIV keeps its reticular structure because of impaired pruning. (F) A graph comparing the SIV vascular loop formation and remodeling in a wild-type (grey) and silent heart embryo (orange), based on SPIM time-lapse experiments between 36 and 84 hpf. From the left, showing the number of cross branches pruned during remodeling phase, the number of cross branches/loops closed via collateral fusion, the number of cross branches/loops remaining until the end of the movie, and the sum of all loops observed throughout the movie. The values are average numbers per SIV plexus with standard deviation (n = 19 for wild type [WT] and n = 9 for silent heart [SIH]). *** p < 0.001. (G) A graph showing the percentage contribution of pruned (grey), closed by collateral fusion (orange), and remaining (blue) vascular loops to all events observed in WT versus silent heart embryos. See also S1 and S2 Figs, S1–S4 Movies, S1 Table and S1 Data.

Mentions: Because a detailed description of SIV formation is missing, we followed the development of the entire plexus over several days by using Selective Plane Illumination Microscopy (SPIM), a novel technique for fast, long-term imaging of large fields of view [16]. In SPIM, images from different angles are acquired by rotating the sample, which is particularly beneficial for the large and curved structure of the SIV. Furthermore, the noninvasive embryo mounting [17] combined with low phototoxicity minimize interference of the imaging with the development of the vasculature [18]. We imaged Tg(fli1a:EGFP)y1 transgenic zebrafish embryos over 48 h (from ~36 to ~84 hours post-fertilization [hpf]) (S1 Movie and S2 Movie and Fig 1A–1E’). Based on these observations, we can divide the development of the SIV into four phases: (I) formation of the primary SIV tube through ventral sprouting of endothelial cells from the PCV followed by cell coalescence (Fig 1A–1A’, see S1A Fig and S3 Movie for details); (II) vascular loop formation through fusion of angiogenic sprouts originating from the primary SIV (Fig 1B–1B’, see S1B Fig and S4 Movie for details); (III) formation of a reticular structure with multiple vascular loops (Fig 1C–1C’); and (IV) remodeling into the final structure with parallel, vertical branches that drain into the large ventral SIV (Fig 1D–1E’).


Endothelial cell self-fusion during vascular pruning.

Lenard A, Daetwyler S, Betz C, Ellertsdottir E, Belting HG, Huisken J, Affolter M - PLoS Biol. (2015)

SIV development and maturation.(A–D) A model representing four phases of SIV plexus development in the zebrafish embryo between ~36 and 84 hpf. The SIV plexus is blue, and the dorsal aorta (DA), posterior cardinal vein (PCV), and intersegmental vessels are marked in grey. Single endothelial cells sprout ventrally and separate from the PCV (A) to form a primary SIV branch (B). Angiogenic sprouts grow out of the primary SIV and fuse to each other, forming a reticular plexus with multiple cross branches (C, red) and vascular loops (C, green); simultaneously, the plexus grows and moves ventrally. Eventually, the cross branches (and hence the loops) are removed, and the plexus simplifies, forming a set of parallel vertical branches draining into a large ventral SIV branch (D). (A’–E’) Stills from a SPIM time-lapse movie representing five phases of SIV development corresponding to models in A. (A”–E”) Stills from a SPIM time-lapse movie representing SIV development in a silent heart morphant embryo corresponding to models in A. In this case, the SIV keeps its reticular structure because of impaired pruning. (F) A graph comparing the SIV vascular loop formation and remodeling in a wild-type (grey) and silent heart embryo (orange), based on SPIM time-lapse experiments between 36 and 84 hpf. From the left, showing the number of cross branches pruned during remodeling phase, the number of cross branches/loops closed via collateral fusion, the number of cross branches/loops remaining until the end of the movie, and the sum of all loops observed throughout the movie. The values are average numbers per SIV plexus with standard deviation (n = 19 for wild type [WT] and n = 9 for silent heart [SIH]). *** p < 0.001. (G) A graph showing the percentage contribution of pruned (grey), closed by collateral fusion (orange), and remaining (blue) vascular loops to all events observed in WT versus silent heart embryos. See also S1 and S2 Figs, S1–S4 Movies, S1 Table and S1 Data.
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Related In: Results  -  Collection

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Show All Figures
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pbio.1002126.g001: SIV development and maturation.(A–D) A model representing four phases of SIV plexus development in the zebrafish embryo between ~36 and 84 hpf. The SIV plexus is blue, and the dorsal aorta (DA), posterior cardinal vein (PCV), and intersegmental vessels are marked in grey. Single endothelial cells sprout ventrally and separate from the PCV (A) to form a primary SIV branch (B). Angiogenic sprouts grow out of the primary SIV and fuse to each other, forming a reticular plexus with multiple cross branches (C, red) and vascular loops (C, green); simultaneously, the plexus grows and moves ventrally. Eventually, the cross branches (and hence the loops) are removed, and the plexus simplifies, forming a set of parallel vertical branches draining into a large ventral SIV branch (D). (A’–E’) Stills from a SPIM time-lapse movie representing five phases of SIV development corresponding to models in A. (A”–E”) Stills from a SPIM time-lapse movie representing SIV development in a silent heart morphant embryo corresponding to models in A. In this case, the SIV keeps its reticular structure because of impaired pruning. (F) A graph comparing the SIV vascular loop formation and remodeling in a wild-type (grey) and silent heart embryo (orange), based on SPIM time-lapse experiments between 36 and 84 hpf. From the left, showing the number of cross branches pruned during remodeling phase, the number of cross branches/loops closed via collateral fusion, the number of cross branches/loops remaining until the end of the movie, and the sum of all loops observed throughout the movie. The values are average numbers per SIV plexus with standard deviation (n = 19 for wild type [WT] and n = 9 for silent heart [SIH]). *** p < 0.001. (G) A graph showing the percentage contribution of pruned (grey), closed by collateral fusion (orange), and remaining (blue) vascular loops to all events observed in WT versus silent heart embryos. See also S1 and S2 Figs, S1–S4 Movies, S1 Table and S1 Data.
Mentions: Because a detailed description of SIV formation is missing, we followed the development of the entire plexus over several days by using Selective Plane Illumination Microscopy (SPIM), a novel technique for fast, long-term imaging of large fields of view [16]. In SPIM, images from different angles are acquired by rotating the sample, which is particularly beneficial for the large and curved structure of the SIV. Furthermore, the noninvasive embryo mounting [17] combined with low phototoxicity minimize interference of the imaging with the development of the vasculature [18]. We imaged Tg(fli1a:EGFP)y1 transgenic zebrafish embryos over 48 h (from ~36 to ~84 hours post-fertilization [hpf]) (S1 Movie and S2 Movie and Fig 1A–1E’). Based on these observations, we can divide the development of the SIV into four phases: (I) formation of the primary SIV tube through ventral sprouting of endothelial cells from the PCV followed by cell coalescence (Fig 1A–1A’, see S1A Fig and S3 Movie for details); (II) vascular loop formation through fusion of angiogenic sprouts originating from the primary SIV (Fig 1B–1B’, see S1B Fig and S4 Movie for details); (III) formation of a reticular structure with multiple vascular loops (Fig 1C–1C’); and (IV) remodeling into the final structure with parallel, vertical branches that drain into the large ventral SIV (Fig 1D–1E’).

Bottom Line: Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail.In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection.Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum der Universität Basel, Basel, Switzerland.

ABSTRACT
During embryonic development, vascular networks remodel to meet the increasing demand of growing tissues for oxygen and nutrients. This is achieved by the pruning of redundant blood vessel segments, which then allows more efficient blood flow patterns. Because of the lack of an in vivo system suitable for high-resolution live imaging, the dynamics of the pruning process have not been described in detail. Here, we present the subintestinal vein (SIV) plexus of the zebrafish embryo as a novel model to study pruning at the cellular level. We show that blood vessel regression is a coordinated process of cell rearrangements involving lumen collapse and cell-cell contact resolution. Interestingly, the cellular rearrangements during pruning resemble endothelial cell behavior during vessel fusion in a reversed order. In pruning segments, endothelial cells first migrate toward opposing sides where they join the parental vascular branches, thus remodeling the multicellular segment into a unicellular connection. Often, the lumen is maintained throughout this process, and transient unicellular tubes form through cell self-fusion. In a second step, the unicellular connection is resolved unilaterally, and the pruning cell rejoins the opposing branch. Thus, we show for the first time that various cellular activities are coordinated to achieve blood vessel pruning and define two different morphogenetic pathways, which are selected by the flow environment.

No MeSH data available.


Related in: MedlinePlus