Within the circulatory system, blood flow regulates vascular remodelling1, stimulates blood stem cell formation2, and has a role in the pathology of vascular disease3. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation4. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system4. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow1. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a5,6,7 induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.
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We would like to thank B. Roman, V. Ambros and C. Sagerstrom for critical review of the manuscript. We thank J.-N. Chen for providing the Tg(kdrl:egfp)la116 zebrafish line. We appreciate the assistance of T. Stork and M. Freeman for help in performing laser assisted microsurgery. We also thank C. Grabher for the gift of gata1 morpholino. We are grateful to M. Beltrame for providing the cdh5 plasmid and T. Smith and S. Sheppard for technical assistance. We thank M. Green and N. Wajapeyee for the Ras-transformed NIH3T3 cell line. This work was supported in part by grants from the National Heart, Lung, and Blood Institute and National Cancer Institute (N.D.L.). The Bioimaging Group (C.S. and K.E.F.) is a core resource supported by a Diabetes Endocrinology Research Center (DERC) grant DK32520 from the National Institute of Diabetes and Digestive and Kidney Diseases. N.D.L and K.E.F are members of the UMass DERC (DK32520). We apologize to researchers whose work we were unable to cite due to space constraints.
Author Contributions S.N. designed and carried out all experiments, analysed data and wrote the paper. C.S. and K.E.F. performed two-photon imaging. P.W. and A.H. developed and provided the miRNA transgenic expression vector. N.D.L constructed the miRNA sensor vector, designed experiments, analysed data and wrote the paper.
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-12 with legends, Supplementary Tables 1-3 and legends for Supplementary Movies 1-11. (PDF 9741 kb)
This movie shows wild type Tg(kdrl:egfp)la116 embryo imaged by 2-photon microscopy (see Supplementary information file for full legend). (MOV 5805 kb)
This movie shows wild type Tg(kdrl:egfp)la116 embryo imaged by 2-photon microscopy (see Supplementary information file for full legend). (MOV 3239 kb)
This file contains a video of aortic arch circulation in a wild type embryo at 57 hpf (see Supplementary information file for full legend). (MOV 6983 kb)
This file contains a video of aortic arch circulation in a wild type embryo at 65 hpf (see Supplementary information file for full legend). (MOV 2992 kb)
This movie shows wild type Tg(kdrl:egfp)la116 embryo treated with BDM beginning at 46 hpf and imaged by 2-photon microscopy (see Supplementary information file for full legend). (MOV 2371 kb)
This movie shows wild type Tg(kdrl:egfp)la116 embryo treated with Tricaine beginning at 46 hpf and imaged by 2-photon microscopy (see Supplementary information file for full legend). (MOV 2010 kb)
This movie shows wild type Tg(fli1a:negfp)y7 embryo (see Supplementary information file for full legend). (MOV 3406 kb)
This movie shows wild type Tg(fli1a:negfp)y7 embryo treated with Tricaine (see Supplementary information file for full legend). (MOV 6062 kb)
This file contains a video of aortic arch circulation in an embryo injected with 11 ng klf2a Morpholino at 65 hpf (see Supplementary information file for full legend). (MOV 2651 kb)
This file contains a video of aortic arch circulation at 60 hpf in an embryo injected with 20 ng of miR-126 Morpholino hpf (see Supplementary information file for full legend). (MOV 4354 kb)
This movie shows Tg(kdrl:egfp)la116 embryo injected with 20 ng of miR-126 Morpholino hpf and imaged by 2-photon microscopy (see Supplementary information file for full legend). (MOV 4248 kb)
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Nicoli, S., Standley, C., Walker, P. et al. MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464, 1196–1200 (2010). https://doi.org/10.1038/nature08889
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