Haematopoietic stem and progenitor cells (HSPCs) give rise to all blood lineages that support the entire lifespan of vertebrates1. After HSPCs emerge from endothelial cells within the developing dorsal aorta, homing allows the nascent cells to anchor in their niches for further expansion and differentiation2,3,4,5. Unique niche microenvironments, composed of various blood vessels as units of microcirculation and other niche components such as stromal cells, regulate this process6,7,8,9. However, the detailed architecture of the microenvironment and the mechanism for the regulation of HSPC homing remain unclear. Here, using advanced live imaging and a cell-labelling system, we perform high-resolution analyses of the HSPC homing in caudal haematopoietic tissue of zebrafish (equivalent to the fetal liver in mammals), and reveal the role of the vascular architecture in the regulation of HSPC retention. We identify a VCAM-1+ macrophage-like niche cell population that patrols the inner surface of the venous plexus, interacts with HSPCs in an ITGA4-dependent manner, and directs HSPC retention. These cells, named ‘usher cells’, together with caudal venous capillaries and plexus, define retention hotspots within the homing microenvironment. Thus, the study provides insights into the mechanism of HSPC homing and reveals the essential role of a VCAM-1+ macrophage population with patrolling behaviour in HSPC retention.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Any Methods, including any statements of data availability and Nature Research reporting summaries, along with any additional references and Source Data files, are available in the online version of the paper. 3D reconstruction of vessel surrounding HSPCs in retention hotspot is deposited at http://www.biosino.org/node/project/detail/OEP000169.
Morrison, S. J., Uchida, N. & Weissman, I. L. The biology of hematopoietic stem cells. Annu. Rev. Cell Dev. Biol. 11, 35–71 (1995).
Murayama, E. et al. Tracing hematopoietic precursor migration to successive hematopoietic organs during zebrafish development. Immunity 25, 963–975 (2006).
Kissa, K. & Herbomel, P.Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464, 112–115 (2010).
Bertrand, J. Y. et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464,108–111 (2010).
Boisset, J. C. et al. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120 (2010).
Xue, Y. et al. The vascular niche regulates hematopoietic stem and progenitor cell lodgment and expansion via klf6a-ccl25b. Dev. Cell 42, 349–362.e4 (2017).
Mahony, C. B., Fish, R. J., Pasche, C. & Bertrand, J. Y. tfec controls the hematopoietic stem cell vascular niche during zebrafish embryogenesis. Blood 128, 1336–1345 (2016).
Glass, T. J. et al. Stromal cell-derived factor-1 and hematopoietic cell homing in an adult zebrafish model of hematopoietic cell transplantation. Blood 118, 766–774 (2011).
Murayama, E. et al. NACA deficiency reveals the crucial role of somite-derived stromal cells in haematopoietic niche formation. Nat. Commun. 6, 8375 (2015).
Jin, H., Xu, J. & Wen, Z. Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development. Blood 109, 5208–5214 (2007).
Burns, C. E., Traver, D., Mayhall, E., Shepard, J. L. & Zon, L. I. Hematopoietic stem cell fate is established by the Notch–Runx pathway. Genes Dev. 19, 2331–2342 (2005).
Jao, L. E., Wente, S. R. & Chen, W. Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system. Proc. Natl Acad. Sci. USA 110, 13904–13909 (2013).
Soza-Ried, C., Hess, I., Netuschil, N., Schorpp, M. & Boehm, T. Essential role of c-myb in definitive hematopoiesis is evolutionarily conserved. Proc. Natl Acad. Sci. USA 107, 17304–17308 (2010).
Tamplin, O. J. et al. Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche. Cell 160, 241–252 (2015).
Arroyo, A. G., Yang, J. T., Rayburn, H. & Hynes, R. O. α4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity 11, 555–566 (1999).
Imai, Y., Shimaoka, M. & Kurokawa, M. Essential roles of VLA-4 in the hematopoietic system. Int. J. Hematol. 91, 569–575 (2010).
Gribi, R., Hook, L., Ure, J. & Medvinsky, A. The differentiation program of embryonic definitive hematopoietic stem cells is largely α4 integrin independent. Blood 108, 501–509 (2006).
Qian, H. et al. Distinct roles of integrins α6 and α4 in homing of fetal liver hematopoietic stem and progenitor cells. Blood 110, 2399–2407 (2007).
Scott, L. M., Priestley, G. V. & Papayannopoulou, T. Deletion of α4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol. Cell. Biol. 23, 9349–9360 (2003).
Nombela-Arrieta, C. et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nat. Cell Biol. 15, 533–543 (2013).
Osborn, L. et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell 59, 1203–1211 (1989).
Elices, M. J. et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell 60, 577–584 (1990).
Koenig, J. M., Ballantyne, C. M., Kumar, A. G., Smith, C. W. & Yoder, M. C. Vascular cell adhesion molecule-1 expression and hematopoietic supportive capacity of immortalized murine stromal cell lines derived from fetal liver and adult bone marrow. In Vitro Cell. Dev. Biol. Anim. 38, 538–543 (2002).
Berlin, C. et al. α4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80, 413–422 (1995).
Warga, R. M., Kane, D. A. & Ho, R. K. Fate mapping embryonic blood in zebrafish: multi- and unipotential lineages are segregated at gastrulation. Dev. Cell 16, 744–755 (2009).
Winkler, I. G. et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood 116, 4815–4828 (2010).
Dutta, P. et al. Macrophages retain hematopoietic stem cells in the spleen via VCAM-1. J. Exp. Med. 212, 497–512 (2015).
Jin, H. et al. Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI. Development 136, 647–654 (2009).
Traver, D. et al. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat. Immunol. 4, 1238–1246 (2003).
Shi, X. et al. Functions of idh1 and its mutation in the regulation of developmental hematopoiesis in zebrafish. Blood 125, 2974–2984 (2015).
Hall, C., Flores, M. V., Storm, T., Crosier, K. & Crosier, P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42 (2007).
Liu, C. et al. Macrophages mediate the repair of brain vascular rupture through direct physical adhesion and mechanical traction. Immunity 44, 1162–1176 (2016).
Cross, L. M., Cook, M. A., Lin, S., Chen, J. N. & Rubinstein, A. L. Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay. Arterioscler. Thromb. Vasc. Biol. 23, 911–912 (2003).
Davison, J. M. et al. Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev. Biol. 304, 811–824 (2007).
Scott, E. K. & Baier, H. The cellular architecture of the larval zebrafish tectum, as revealed by Gal4 enhancer trap lines. Front. Neural Circuits 3, 13 (2009).
Moro, E. et al. In vivo Wnt signaling tracing through a transgenic biosensor fish reveals novel activity domains. Dev. Biol. 366, 327–340 (2012).
Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. & Lieschke, G. J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49–e56 (2011).
Bahary, N. et al. The Zon laboratory guide to positional cloning in zebrafish. Methods Cell Biol. 77, 305–329 (2004).
Knapik, E. W. et al. A microsatellite genetic linkage map for zebrafish (Danio rerio). Nat. Genet. 18, 338–343 (1998).
Scheer, N. & Campos-Ortega, J. A. Use of the Gal4-UAS technique for targeted gene expression in the zebrafish. Mech. Dev. 80, 153–158 (1999).
Lam, E. Y. N. et al. Zebrafish runx1 promoter–EGFP transgenics mark discrete sites of definitive blood progenitors. Blood 113, 1241–1249 (2009).
Nasevicius, A. & Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216–220 (2000).
Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K. & Kawakami, K. Transgenesis in zebrafish with the Tol2 transposon system. Methods Mol. Biol. 561, 41–63 (2009).
Xiao, A. et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res. 41, e141 (2013).
Gao, L. et al. TopBP1 governs hematopoietic stem/progenitor cells survival in zebrafish definitive hematopoiesis. PLoS Genet. 11, e1005346 (2015).
Patterson, L. J. et al. The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish. Blood 109, 2389–2398 (2007).
Jia, X. E. et al. Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction. Cell Res. 25, 946–962 (2015).
Zakrzewska, A. et al. Macrophage-specific gene functions in Spi1-directed innate immunity. Blood 116, e1–e11 (2010).
Jowett, T. & Lettice, L. Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes. TIG 10, 73–74 (1994).
Wang, H. et al. Dual-color ultrasensitive bright-field RNA in situ hybridization with RNAscope. Methods Mol. Biol. 1211, 139–149 (2014).
Murphey, R. D., Stern, H. M., Straub, C. T. & Zon, L. I.A chemical genetic screen for cell cycle inhibitors in zebrafish embryos. Chem. Biol. Drug Des. 68, 213–219 (2006).
Mazzocco, C. et al. In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe. Sci. Rep. 6, 23314 (2016).
Curado, S. et al. Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies. Dev. Dyn. 236, 1025–1035 (2007).
Chudakov, D. M., Lukyanov, S. & Lukyanov, K. A. Using photoactivatable fluorescent protein Dendra2 to track protein movement. Biotechniques 42, https://doi.org/10.2144/000112470 (2007).
Chudakov, D. M., Lukyanov, S. & Lukyanov, K. A. Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2. Nat. Protocols 2, 2024–2032 (2007).
Chen, Q. et al. Haemodynamics-driven developmental pruning of brain vasculature in zebrafish. PLoS Biol. 10, e1001374 (2012).
Kamoun, W. S. et al. Simultaneous measurement of RBC velocity, flux, hematocrit and shear rate in vascular networks. Nat. Methods 7, 655–660 (2010).
Schaffer, C. B. et al. Two-photon imaging of cortical surface microvessels reveals a robust redistribution in blood flow after vascular occlusion. PLoS Biol. 4, e22 (2006).
We thank the following people for the zebrafish transgenic lines: L. Luo for Tg(kdrl:Dendra2), Z. Wen for Tg(mpeg1:Gal4,UAS:NfsB-mCherry), Tg(UAS:Kaede) and Tg(mpeg1:eGFP), B. Blazar for Tg(cxcl12a:dsRed) and F. Argenton for Tg(tcf:eGFP). We are also grateful to M. Deng and J. He for technical support, and Z. Wen, L. Li, L. Zon, J. Peng and A. Meng for discussions. This work was granted by CAS Strategic Priority Research Program (XDB19030000), Ministry of Science and Technology of China (2017YF0503600), National Natural Science Foundation of China (31571505 and 31371461), CAS Scientific Research Equipment Development Project (YZ201646) and Science and Technology Commission of Shanghai Municipality (13JC1406400) to W.J.P.
Nature thanks P. Herbomel and the anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Normal primitive haematopoiesis is intact in mutcas005. WISH results demonstrate that the expression of primitive haematopoietic cell markers is identical between siblings and mutcas005 embryos at 22 h.p.f., including scl (also known as tal1; haematopoietic progenitor marker), gata1 (also known as gata1a; erythrocyte progenitor marker) and pu.1 (also known as spi1b; myeloid progenitor marker). b, The vascular development is normal in mutcas005 embryos. WISH results show no difference in the expression of kdrl (pan-endothelial cell marker) at 36 h.p.f. between wild-type siblings and mutcas005 embryos. c, The haemogenic endothelium is intact in mutcas005 embryos. WISH results show no difference in runx1 expression at 36 h.p.f. in wild-type and mutcas005 embryos. d, e, The definitive haematopoiesis is defective in zebrafish mutcas005 embryos. d, Bright-field images of wild-type and mutcas005 embryos show no obvious morphological difference at 5 d.p.f. e, WISH results of myb, hbae1.1, mpx and lyz expression in wild-type and mutcas005 embryos at 5 d.p.f. Arrows indicate the comparable position in wild-type (black) or mutcas005 embryos (red). In 5 d.p.f. wild-type embryos, myb was expressed in all haematopoietic tissues including the CHT, thymus and kidney, whereas homozygous mutcas005 embryos displayed markedly decreased myb expression in the CHT, but similar expression to wild-type embryos in the thymus and kidney marrow. In accordance, the expression of downstream haematopoietic lineage cell markers, including hbae1.1 (erythrocyte marker), mpx (granulocyte marker) and lyz (macrophage marker), also showed similar expression patterns in the wild-type (black) and mutcas005 (red) embryos to that of the myb WISH analysis. f, The percentage of apoptotic HSPCs detected by the TUNEL assay in 2 and 4 d.p.f. wild-type and mutcas005 embryos. 2 d.p.f.: P = 0.35, t = 0.96, df = 27; 4 d.p.f.: P = 0.50, t = 0.69, df = 27. Error bars denote s.e.m. g, At 7 d.p.f., myb expression in the thymus and kidney marrow was identical in wild-type sibling and mutcas005 embryos, whereas mutcas005 embryos still displayed markedly decreased myb expression in the CHT. In addition, mutcas005 embryos displayed notably decreased hbae1.1, mpx and lyz expression in the CHT but not in the kidney marrow.
a, Positional cloning of mutcas005. After high-resolution mapping, the point mutation is flanked by the SSLP markers z8363 (5 recombinant out of 994 meiosis) and zK165L22 (1 recombinant out of 994 meiosis). This region contains the four genes cwc22, ube2e3, itga4 and cerkl. The red strip represents chromosome 9; the positions and recombination of the SSLP markers are indicated. SSLP markers on the same side of the mutation site are shown in the same colour. b, Generation of itga4 mutants via the ENU(top) or CRISPR–Cas9 (bottom) technique. The alignment of wild-type (underlined) and mutated sequences is listed. The insertion in ENU is indicated in red (an insertion of G leading to an earlier stop codon in the itga4 gene in mutcas005). The PAM sequence of gRNA is ‘CGG’ (blue). Deletions are indicated by dashes. c, According to the stop codon in the genome of itga4 mutants, SMART software was used to predict the structure of the wild-type itga4, itga4cas005 and itga4cas010 presumed protein. The molecular sizes of the presumed protein are indicated. d, The myb WISH results in wild-type and itga4cas010 embryos at 72 h.p.f. e, itga4 morphants could phenocopy itga4cas005. The validated itga4 morpholino oligonucleotide (MO) that can block the translation of itga4 mRNA was injected into one-cell-stage wild-type embryos to produce itga4 morphants. WISH results of myb expression in the control and itga4 morphants at 72 h.p.f. f, g, Representative live imaging (g) and statistical analysis (f) of Tg(gata1:DsRed), Tg(mpx:eGFP) and Tg(lyz:DsRed) transgenic embryos after the injection of control or itga4 or vcam1 morpholino oligonucleotides at 84 h.p.f. Results show that downstream haematopoietic lineages were all defective owing to disrupted HSPC retention in the CHT. gata1, control vs itga4 MO: ****P < 0.0001, t = 7.42, df = 14; gata1, control vs vcam1 MO: ****P < 0.0001, t = 8.11, df = 14; mpx, control vs itga4 MO: P < 0.0001, t = 5.92, df = 14; mpx, control vs vcam1 MO: ****P < 0.0001, t = 9.41, df = 14; lyz, control vs itga4 MO: ****P < 0.0001, t = 7.16, df = 25; lyz, control vs vcam1 MO: ****P < 0.0001, t = 5.74, df = 25. h, In situ analysis of itga4 expression in the AGM (FISH, 36 h.p.f.) and CHT (WISH, 72 h.p.f.) of Tg(kdrl:eGFP) (green) embryos after injection of control, runx1 or myb morpholino oligonucleotides indicates HSPC cell-autonomous expression. i–k, itga4 has an HSPC intrinsic mechanism during definitive haematopoiesis. i, The construction of the plasmid that was applied in the itga4cas005 mutant for Tol2-transposase-mediated transient transgenesis of runx1-enhancer-driven wild-type itga4 expression. j, k, Phenotype analysis by myb WISH shows that the construct had a notable rescue effect on definitive haematopoiesis in the itga4cas005 mutant at 72 h.p.f. More than 45% of mCherry+ cells overlapped with myb FISH signalling in the CHT; a representative image is shown in k. Error bars denote s.e.m. Scale bars, 50 μm (g), 20 μm (h) and 5 μm (k).
Extended Data Fig. 3 Photoconversion of Tg(kdrl:Dendra2) cells in the AGM can specifically mark HSPCs in the CHT.
a, Schematic illustration (left) and confocal imaging analysis (right) of the HSPC labelling system in live Tg(kdrl:Dendra2) transgenic zebrafish larva. At 32–36 h.p.f., an ultraviolet laser was applied to photoconvert Dendra2 in haemogenic endothelium from green to red fluorescence in the area marked by the rectangle. Endothelial-to-haematopoietic transition was observed with egress of single red Dendra2+ cells (white arrows) from the aortic ventral wall into the sub-aortic space. b, Flow chart of the experimental analysis on HSPCs (red Dendra2+ cells) in live-imaging and confocal-imaging analysis after in situ RNA scope in identical locations. Green Dendra2 signalling could remain during the experiment, whereas red Dendra2 signalling could not (the red fluorescence of the ISV disappeared after in situ RNA scope analysis). Photoconverting the ISV in different embryos makes each embryo distinguishable. (We distinguished the embryos by the position of the photoconverted and weakened Dendra2 green ISV). After live imaging the red Dendra2+ cells in the CHT (top, arrows) at 54 h.p.f., the embryos were fixed immediately for runx1 in situ RNA scope analysis (bottom, yellow arrowheads). All HSPCs (red Dendra2+ cells) carry runx1 transcripts, whereas there was no signal in the negative control. c, d, Confocal images of the CHT (c) and statistical analysis (d) at 54 h.p.f. show markedly reduced numbers of HSPCs (red Dendra2+ cells) in runx1 and myb morphants, compared to that in control morphants. Control vs runx1 MO: ****P < 0.0001, t = 9.15, df = 9; control vs myb MO: ****P < 0.0001, t = 8.66, df = 9. e, Live-imaging analysis on individual HSPCs in wild-type and itga4cas005 mutant embryos. Three representative frames from time-lapse imaging of 52–60 h.p.f. wild-type and itga4cas005 embryos with the Tg(kdrl:Dendra2) labelling system. The HSPCs (white arrow) remained stable in the vascular niche for over 30 min in the wild-type sibling (left); however, in the itga4cas005 mutants (right), the HSPCs (yellow arrowhead) remained for less than 9 min (see Supplementary Video 2). Error bars represent s.e.m. Scale bars, 50 μm (a–c) and 20 μm (e).
a, Correlation analysis of retention time and the dorsal–ventral relative location of individual HSPCs in the CHT of four wild-type embryos at 50–60 h.p.f. Each shape represents one embryo (triangle, circle, square and rhombus), and each colour represent one class of retention time zone. HSPCs that remained for longer than 30 min were preferentially located in the region between the two magenta dashed lines enriched with venous capillaries. b–d, Longitudinal whole-mount images of the CHT in wild-type siblings (b, c) and itga4cas010 mutant embryos (d) in a Tg(kdrl:Dendra2) background after photoconversion and retention calculation of the frequency of HSPC appearance. e, Longitudinal whole-mount images of the CHT in Tg(mpeg1:Gal4,kdrl:Dendra2) embryos after MTZ treatment with or without a Tg(UAS:NfsB-mCherry) background, and vcam1cas011; Tg(mpeg1:Gal4,kdrl:Dendra2) embryos with transient transgenesis of vector (UAS:polyA) or UAS:vcam1 after photoconversion and retention calculation of the frequency of HSPC appearance. Red arrows denote retention hotspots. HSPCs were preferentially located in the region between the two magenta dashed lines. Retention hotspots disappeared in the itga4cas010and vcam1cas011 mutants after depletion of macrophages (MTZ treatment on NfsB-expressing macrophages). Scale bars, 50 μm.
a, Representative spatial maps of the flow velocity of photoconverted HSPCs in the caudal vasculature at 54 h.p.f. Arrows show the direction of blood flow, and different colours indicate the level of velocity. b, Schematic illustration of how HSPCs enter the CHT via the circulation. c–e, Time-lapse imaging (c, d) and speed curve diagram (e) show that HSPCs arrive either from the intersegmental vessel (green arrow in c) or the CVP (red arrowhead in d) into the CHT and gradually decelerate (see Supplementary Video 3). Scale bar, 20 μm.
Extended Data Fig. 6 The representative high-resolution vascular structure and HSPC in the retention hotspot.
a, The original fluorescent image of the vessel surrounding HSPCs in the retention hotspot was captured by an LSM880 microscope equipped with Airyscan function and processed by 3D reconstruction (see Supplementary Video 4). b, 3D reconstruction of a. c, Section view of the caudal vein plexus and capillary. d, The 45° rotation view of the red frame in b. e, Time-lapse imaging (left) and scheme graph (right) show how HSPC retention occurs. HSPCs initially came into the venous plexus and then entered the venous capillary for long-term retention. f–h, Images (f, g) and statistical analysis (h) show that the diameter of various vessels in the CHT in itga4cas010 mutants (g) is similar to that in the wild-type siblings (f) at 54 h.p.f. The inner diameter of venous capillaries, but not other vessels, is close to the diameter of HSPCs. DA: P = 0.73, t = 0.35, df = 19; CV: P = 0.14, t = 1.52, df = 33; CVP: P = 0.17, t = 1.41, df = 19; VC: P = 0.63, t = 0.49, df = 19; HSPC: P = 0.67, t = 0.44, df = 19. Scale bar, 20 μm.
a, Generation of the vcam1 mutant using the CRISPR–Cas9 technique. The alignment of wild-type (underlined) and mutated sequences is listed. The PAM sequence of gRNA is ‘GGG’ (in blue). Deletions are indicated by dashes. b, According to the stop codon in the genome, SMART software was used to predict the structure of the wild-type vcam1 and vcam1cas011 presumed protein. The molecular sizes of the presumed protein are indicated. c, Live imaging of the CHT at 54 h.p.f. shows retention defects in vcam1cas011 mutants. Representative images show that most HSPCs resided within the CHT (white arrows) in wild-type siblings (top), whereas these cells went through quickly in vcam1cas011 embryos (bottom, yellow arrowheads) (see Supplementary Video 5). d, WISH analysis of myb expression in the CHT of wild-type and vcam1cas011 embryos at 72 h.p.f. e, f, The definitive haematopoiesis is defective in vcam1cas011 mutant zebrafish embryos. e, The bright-field images of wild-type and vcam1cas011 embryos show no obvious morphological difference at 72 h.p.f. f, WISH results of hbae1.1, mpx and lyz expression in wild-type and vcam1cas011mutant embryos at 72 h.p.f. Arrows indicate the comparable position in wild-type (black) or vcam1cas011 (red) embryos. g, Magnified views showed VCAM-1 was mainly expressed in individual cells (white arrow) but weakly expressed on the venous endothelial cells (yellow arrowheads). h, After photoconversion, Tg(kdrl:Dendra2) embryos are stained with anti-VCAM-1 (magenta, white arrow). The yellow arrowhead denotes an HSPC. i, Tg(cxcl12a:DsRed,kdrl:eGFP) transgenic embryos are stained with anti-VCAM-1 (magenta, white arrow) and anti-DsRed (red, yellow arrowhead). j, Tg(tcf:eGFP,kdrl:mCherry) transgenic embryos are stained with anti-VCAM-1 (magenta, white arrows) and anti-GFP (green, yellow arrowheads). Scale bars, 50 μm (c), 20 μm (g) and 10 μm (h, i).
a, Representative FISH confocal imaging of mfap4 (top), csf1ra (middle), spi1a (bottom) immunofluorescence with anti-VCAM-1 and anti-GFP antibodies indicates that VCAM-1+ cells in the CHT are macrophage-like cells (see Supplementary Table 2). b, The construction of the plasmid applied in c–f. c, Validation of the macrophage-specific cell-depletion system. Left, the number of HSPCs under the endothelial-to-haematopoietic transition (EHT) process in the AGM of Tg(mpeg1:Gal4,kdrl:Dendra2) transgenic embryos at 54 h.p.f. with MTZ treatment and with (#2) or without (#1) the Tg(UAS:NfsB-mCherry) background. P = 0.80, t = 0.25, df = 9. Right, live imaging of vessels (green) and macrophages (red) with or without MTZ treatment in the CHT of Tg(kdrl:Dendra2,mpeg1:Gal4,UAS:NfsB-mCherry) transgenic embryos showed that MTZ treatment could delete almost all mCherry+ macrophages. d, Quantification of VCAM-1+ cells in the CHT, detected by anti-VCAM-1 immunofluorescence, in Tg(mpeg1:Gal4,kdrl:Dendra2) embryos at 54 h.p.f. with MTZ treatment and with (#2) or without (#1) a Tg(UAS:NfsB-mCherry) background, and in vcam1cas011 mutants with Tol2-mediated transient transgenesis of vector (UAS:polyA) (#3) or UAS:vcam1 (#4) in a Tg(mpeg1:Gal4,kdrl:Dendra2) background. #1 vs #2: ****P < 0.0001, t = 9.18, df = 24; #3 vs #4: ****P < 0.0001, t = 10.03, df = 23. e, Statistical analysis shows the percentage of the three types of definitive haematopoietic phenotype (extremely low, decreased or normal) in the four experimental conditions (#1–#4) linked to d. Macrophage-specific cell depletion caused deficient definitive haematopoiesis; however, macrophage-specific VCAM-1 re-expression markedly rescued deficient haematopoiesis in vcam1cas011 mutants. f, Retention time of individual HSPCs in transgenic Tg(mpeg1:Gal4,kdrl:Dendra2) embryos at 50–60 h.p.f. with MTZ treatment and with (#2) or without (#1) a Tg(UAS:NfsB-mCherry) background, and in vcam1cas011 mutants with Tol2-mediated transient transgenesis of vector (UAS:polyA) (#3) or UAS:vcam1 (#4) in a Tg(mpeg1:Gal4,kdrl:Dendra2) background (see Fig. 2f, g). #1 vs #2: ***P = 0.0001, t = 3.85, df = 550; #1 vs #3: ****P < 0.0001, t = 6.05, df = 565; #3 vs #4: ****P < 0.0001, t = 4.37, df = 590. g, Live-imaging frame shots of HSPCs in which macrophage-specific VCAM-1 was in re-expressed vcam1cas011 mutants from Fig. 2g (see Supplementary Video 6). h, Schematic illustration shows that macrophage labelling (photoconverted Kaede+; red) was performed at 18 h.p.f. in the rostral blood island (RBI) in Tg(mpeg1:Gal4,UAS:Kaede) embryos, followed by a 1 nl anti-VCAM-1647 (0.4 ng) antibody injection at 50 h.p.f. Cell-lineage tracing of the labelled macrophages (red; yellow arrowheads) in vivo was performed from 2 h after the injection. Representative images show that macrophages from the rostral blood island at 18 h.p.f. migrate to the CHT, and are VCAM-1+. Scale bars, 50 μm (c), 20 μm (g), 10 μm (h) and 5 μm (a).
Extended Data Fig. 9 Anti-VCAM-1647 antibody labels usher cells without disrupting definitive haematopoiesis.
a, Injection of 1 nl (0.4 ng) of anti-VCAM-1647 antibody labels usher cells (arrows) in wild-type and itga4cas010 mutants in the Tg(kdrl:eGFP) background, whereas injection of either control (non-specific) IgG647 antibody into wild-type cells or anti-VCAM-1647 antibody into vcam1cas011 mutants in the Tg(kdrl:eGFP) background did not label any cells in the retention hotspots. Asterisk indicates nonspecific labelling on a chromatophore in the CHT. b, Anti-VCAM-1647 injection marginally influence definitive haematopoiesis. Statistical analysis shows the percentage of the three types of definitive haematopoietic phenotype in nine different conditions, including wild-type embryos without injection (#1), with 10 nl vehicle (#2) or 10 nl 0.4 mg ml−1 IgG647 injection (#3), itga4cas005 mutants (#4) or vcam1cas011 mutants (#5) without injection, and wild-type embryos with 1–10 nl 0.4 mg ml−1 anti-VCAM-1647 injection (#6–#9). c, d, Live imaging of HSPCs (c) or WISH analysis of the myb probe at 60 h.p.f. (d) of the wild-type CHT after vehicle or 1 nl anti-VCAM-1647 antibody injection. e, Schematic diagrams (left) and confocal imaging (right) show VCAM-1+ cells patrolling on a small scale in the CHT in itga4cas010 mutant embryos. Cross indicates the original position at the initial time point. f, Statistical analysis of the duration of the interaction between HSPCs and usher cells, the HSPC retention time, the diameter of vessels and the dorsal–ventral relative location for HSPC retention in pre-type I (type 0), pre-type II (type 0), type I and type II. Duration, pre-I vs pre-II: ***P = 0.0001, t = 4.25, df = 41; duration, pre-I vs I: ****P < 0.0001, t = 5.31, df = 43; duration, pre-II vs II: P = 0.37, t = 0.93, df = 12; duration, I vs II: P = 0.46, t = 0.76, df = 14; retention: P = 0.16, t = 1.50, df = 14; diameter, pre-I vs pre-II: ****P < 0.0001, t = 8.80, df = 41; diameter, pre-I vs I: P = 0.68, t = 0.42, df = 43; diameter, pre-II vs II: P = 0.89, t = 0.14, df = 12; diameter, I vs II: **P = 0.006, t = 3.24, df = 14; location, pre-I vs pre-II: ****P < 0.0001, t = 7.64, df = 41; location, pre-I vs I: P = 0.6, t = 0.53, df = 43; location, pre-II vs II: **P = 0.005, t = 3.41, df = 12; location, I vs II: P = 0.41, t = 0.85, df = 14. g, In itga4cas010 mutants, HSPCs encountered but failed to interact with usher cells and then went through the CHT within a few minutes (see Supplementary Video 10). h, The percentage of the type 0, type I and type II HSPC retention types in wild-type sibling and vcam1cas011 mutants in the Tg(mpeg1:Gal4,kdrl:Dendra2) background with transient transgenesis of UAS:vcam1. None of the HSPCs in the vcam1 mutants could be classified into either type I or II retention types, or were comparable with the HSPCs in Extended Data Fig. 8h. Scale bars, 50 μm (a, c) and 20 μm (e, g).
List of genetic cloning and sequencing oligos, morpholino, guide RNA for Cas9-mediated mutagenesis, and primers for cas005 mutant identification or plasmid construction applied in this study.
List of statistic correlation analysis on marker gene expression in mpeg1+ cells and VCAM-1+ cells.
List of parameters on 1) the duration and location of each HSPC-Usher cell interaction and 2) the duration and type of vessel (diameter) for each HSPC retention event.
Macro view of HSPCs homing to the CHT. CHT vasculature (Dendra2+ green) and HSPCs (photoconverted Dendra2+, red) were imaged from 50 hpf for 5 hours in WT sibling and itga4cas005 mutants. HSPCs in itga4 mutants failed in CHT retention, compared to that in wildtype siblings (Link to Fig. 1a). Time-lapse live imaging was captured at 3 minutes/frame and rendered at 5 frames/s. Scale bar, 50 μm.
Micro view of HSPCs homing to vascular niche. CHT vasculature (Dendra2+ green) and HSPCs (photoconverted Dendra2+, red) were imaged with confocal microscope. HSPC in wildtype sibling remained stable in the niche for more than 30 minutes (Left), while in itga4cas005 HSPC stayed less than 9 minutes (Right)(Link to Ex Fig. 3e). Time-lapse live imaging was captured at 3 minutes/frame and rendered at 1 frame/s. Scale bar, 10 μm.
Entries for HSPCs homing to the CHT. HSPCs (photoconverted-Dendra2+, red), in the circulation, entered the CHT vasculature (green) either from ISV or from CVP, and then decelerated in the upper side of CVP (Link to Ex Fig. 5b-e). Time-lapse live imaging was captured at 3 minutes/frame and rendered at 3 frames/s. Scale bar, 20 μm.
3D View of HSPC-vascular niche interaction in a retention hotspot. The high-resolution 3D structure of retention hotspot, including vasculature (Dendra2+, green) and HSPCs (photoconverted Dendra2+, red) were captured by Zeiss 880 confocal microscope equipped with Airyscan module at 56-58 hpf. HSPCs remained in a venous capillary at the upper side of the CVP (Link to Ex Fig. 6a-d). Scale bar, 10 μm.
Defective HSPCs retention in the CHT of vcam-1cas011 mutant. CHT vasculature (Dendra2+, green) and HSPCs (photoconverted Dendra2+, red) were imaged from 50 hpf for 5 hours in vcam-1cas011 mutant. The HSPCs (red) in vcam-1cas011 mutant failed to lodge in the CHT as itga4 mutants (Link to Ex Fig. 7c). Time-lapse live imaging was captured at 3 minutes/frame and rendered at 5 frames/s. Scale bar, 50 μm.
Distinct role of VCAM-1 in macrophages and endothelium in HSPCs retention. HSPCs (photoconverted-Dendra2+, red) labeling was performed at 36 hpf on Tg (kdrl: Dendra2) embryos, with indicated transgenesis or chemical treatment on, positive control (#1, Tg (mpeg1: Gal4; kdrl: Dendra2) embryos treated with MTZ), macrophage-specific cell depletion (#2, Tg (mpeg1: Gal4; UAS: NfsB-mCherry; kdrl: Dendra2) embryos treated with MTZ), negative control (#3, Tg (mpeg1: Gal4; kdrl: Dendra2) embryos in vcam-1cas011 mutant background with vector (UAS: polyA) transient transgenesis), and macrophage-specific VCAM-1 rescue (#4, Tg (mpeg1: Gal4; kdrl: Dendra2) embryos in vcam-1cas011 mutant background with UAS: VCAM-1 transient transgenesis)(Link to Fig. 2e–g, Ex Fig. 8g). Time-lapse live imaging was captured at 5 minutes/frame and rendered at 8 frames/s, followed with HSPC’s behavior analysis and comparison from 54 hpf for 6 hours. Scale bar, 20 μm.
VCAM-1+ Usher cell patrols in the dorsal venous plexus in the CHT. Tg (kdrl: Dendra2) zebrafish larvas were injected with 1 nl (0.4 ng) anti-VCAM-1647 antibody at 50 hpf, following with live imaging from 52 hpf. The VCAM-1+ Usher cell (magenta) is patrolling inside of dorsal venous plexus in the CHT independent of flow direction (Link to Fig. 3c). Time-lapse live imaging was captured at 2.5 minutes/frame and rendered at 1 frame/s. Scale bar, 10 μm.
VCAM-1+ Usher cell guides HSPC into vascular niche. HSPCs (photoconverted-Dendra2+, red) labeling was performed at 36 hpf on Tg (kdrl: Dendra2) embryos, following with 1 nl (0.4 ng) anti-VCAM-1647 antibody injection at 50 hpf. Detection of the interaction between HSPCs (red) and VCAM-1+ cells (magenta) in vivo was acquired from 2 hours post injection. Several representative time-lapse videos show how VCAM-1+ Usher cell-mediated HSPCs retention occurs in the CHT vasculature. VCAM-1+ cells directed HSPC into vascular pocket formed by venous capillaries (Link to Fig. 4a). Time-lapse live imaging was captured at 3 minutes-20 seconds/frame and rendered at 1 frame/s. Scale bar, 10 μm.
Endothelium cuddling occurs upon HSPC arrival. Several representative time-lapse videos show how VCAM-1+ Usher cell-mediated HSPCs retention occurs in the CHT vasculature. HSPC was surrounded by endothelial cells (termed ECs Cuddling) and remained for more than 2 hours (Link to Fig. 4a and b). Time-lapse live imaging was captured at 3 minute-20 seconds/frame and rendered at 1 frame/s. Scale bar, 10 microns.
VCAM-1+ Usher cell fails to interact with itga4-deficient HSPC. Time-lapse imaging analysis on the interaction between HSPCs (red) and VCAM-1+ cells (magenta) in itga4cas010 mutant was acquired 2 hours post injection of anti-VCAM-1647 antibody at 50 hpf. HSPC encountered, but failed to interact with VCAM-1+ Usher cells in itga4 cas010 mutant (Link to Ex Fig. 9g). Time-lapse live imaging was captured at 1.5 minutes /frame, and rendered at 1 frame/s. Scale bar, 10 microns.
About this article
Cite this article
Li, D., Xue, W., Li, M. et al. VCAM-1+ macrophages guide the homing of HSPCs to a vascular niche. Nature 564, 119–124 (2018). https://doi.org/10.1038/s41586-018-0709-7
- Vascular Niche
- Capillary Venules
- Whole-mount In Situ Hybridization (WISH)
- WISH Analysis
- Zeiss LSM510 Confocal Microscope
Clinical Reviews in Allergy & Immunology (2021)
Journal of Hematology & Oncology (2020)
Nature Biotechnology (2020)
Cell Research (2019)