Primitive macrophages control HSPC mobilization and definitive haematopoiesis

In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta–gonad–mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis. Haematopoietic stem/progenitor cells (HSPCs) transform from aortic endothelium into migratory cells that move through stroma and enter circulation to colonize haematopoietic tissues. Here the authors show that HSPCs' passage is facilitated by primitive macrophages that secrete extracellular matrix-degrading enzymes.

In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonadmesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis. B lood cells are continuously produced from self-renewing progenitors with multi-lineage differentiation potential, called haematopoietic stem/progenitor cells (HSPCs). In vertebrates, haematopoiesis occurs as two waves. The 'primitive' wave produces only myelo-erythroid cells, whereas the 'definitive' wave gives rise to HSPCs [1][2][3][4] . In zebrafish, from 16 hours post fertilization (h.p.f.), primitive haematopoietic cells produce macrophages and neutrophils that initiate innate immunity [5][6][7] . Zebrafish first HSPCs appear in the aorta-gonad-mesonephros (AGM), between the dorsal aorta (DA) and the posterior cardinal vein (PCV) by 30 h.p.f. (refs 3,8) where they start to express cd41 marker 9 . These HSPCs originate from aortic endothelial cells 10,11 through a process called endothelial haematopoietic transition or EHT 11 . Live imaging of mouse embryo sections 12 , cell sorting and differentiation experiments in human embryo have shown haemogenic endothelial cells to be at the immediate origin of haematopoietic cell clusters sprouting from the ventral side of the aorta 13 , which suggest that this process is conserved throughout evolution. We have previously documented that zebrafish HSPCs migrate from the AGM, enter the circulation and colonize the caudal haematopoietic tissue (CHT), analogous to the mammalian fetal liver. There they proliferate before colonizing the definitive haematopoietic organs, thymus and kidney 9 . HSPCs emerging between 30 and 55 h.p.f. give rise to all mature blood cell populations, including the second wave of macrophages.
Macrophages from the first and second waves of haematopoiesis play important roles in development, tissue homeostasis 14 , innate and adaptive immunity and wound healing. In adults, they are also involved in the pathologies including chronic diseases 15 and cancer metastasis 16,17 .
Macrophage-related processes are associated with their ability to migrate throughout the body and remodel surrounding stroma 18 by degrading the extracellular matrix (ECM) via the secretion of matrix metalloproteinases (MMPs for human and Mmps for zebrafish) and cathepsins 19 . MMPs have been shown to play important roles in tissue remodelling, or pathological processes 20 , especially MMP-2, 9 and 13. In zebrafish embryos, Mmp-9 and 13a are expressed by myeloid cells from 19 h.p.f. (refs 21,22).
In this study, we imaged the accumulation of primitive macrophages in the AGM of both human and zebrafish embryos and analysed their interactions with emerging HSPCs. We demonstrated that primitive macrophages play a major role in the establishment of definitive haematopoiesis by permitting HSPC mobilization and colonization of haematopoietic organs via Mmps secretion and ECM remodelling.

Results
Macrophages accumulate in AGM during HSPC emergence. Human HSPCs emerge between the 27th and the 40th day of development in the AGM as clusters of CD34 þ CD45 þ cells adhering to the ventral wall of the DA 23 . Immunohistochemistry in human embryos at 34 days of development (n ¼ 2) showed an intriguing accumulation of CD68 þ macrophages in the mesenchyme beneath the ventral part of the CD34 þ aortic endothelium ( Fig. 1a-a 00 , b-b 00 ) and in the HSPC containing intra-aortic clusters (Fig. 1b 0 ,b 00 ). The number of macrophages in these locations, increased between 26 and 37 days of gestation, that is, in concomitance with the emergence of HSPCs (Supplementary Fig. 1; n ¼ 3).
We took advantage of zebrafish embryo remarkable optical properties to study the possible role of these macrophages during definitive haematopoiesis.
In vivo imaging of Tg(cd41:eGFP//mpeg1:mCherry) zebrafish embryos at 43 h.p.f. (green HSPCs and red macrophages) showed a similar accumulation of primitive macrophages along the entire length of the trunk in the direct vicinity of the ventral wall of the DA (Fig. 1c).
Macrophage quantification confirmed their tissue-restricted accumulation in zebrafish AGM compared to surrounding tissues (Fig. 1d,  Macrophages actively patrol in the AGM. To assess if macrophages play a role in HSPC emergence, we imaged the behaviour of HSPCs and macrophages in Tg(cd41:eGFP//mpeg1:mCherry) zebrafish embryos at 43 h.p.f. (Fig. 1e-l). As previously described, endothelial cells from the ventral wall of the aorta first bend then egress into the subaortic space to become free HSPCs 11 . At this stage, we observed that macrophages (Fig. 1f) continuously patrolled between the DA and the PCV, and intimately interacted with emerging HSPCs/cd41:eGFP þ ( Fig. 1e-l, Supplementary Movies 1 and 2). Live imaging of Tg(cd41:eGFP//mpeg1:mCherry) zebrafish embryo at 55 h.p.f. showed a macrophage/ mpeg1:mCherry þ migrating in the AGM around an HSPC/ cd41:eGFP þ during 32 min, before joining the PCV (Supplementary Movie 2, time code 00:56 to 1:00). This HSPC/ cd41:eGFP þ would then join the PCV following the same route (Supplementary Movie 2). A quantification of 112 HSPCs/ cd41:eGFP þ (from 12 embryos) in the AGM revealed that 95.6% of them directly interacted with macrophages suggesting that macrophages could play an important role in emergence of HSPCs in the AGM. Macrophages are essential for definitive haematopoiesis. To characterize their role in HSPC emergence, we chemically and genetically depleted primitive macrophages before or during definitive haematopoiesis at 25 and 45 h.p.f., respectively (before maturation of the second wave of macrophages).
Liposome encapsulated clodronate (L-clodronate) has been widely used to specifically induce the death of phagocytic macrophages in vitro and in vivo in mice and recently in zebrafish 24,25 . We first set about testing its efficiency, toxicity and specificity. Injection of 5 nl of L-clodronate stock suspension into the vein was sufficient for thorough macrophage depletion in embryos between 25  To evaluate the effect of macrophage depletion on HSPC emergence (Fig. 2a-d), we injected L-clodronate in Tg(cd41:eGFP) embryos at 25 h.p.f. and observed that macrophage depletion did not prevent HSPCs/cd41:eGFP þ emergence in the AGM ( Fig. 2b versus a), but led instead to their accumulation at 48 h.p.f. (Fig. 2n, þ 60%, n ¼ 20). Whole-mount in situ hybridization (WISH) of c-myb confirmed the unaffected HSPC emergence in the AGM (Fig. 2d versus c). By contrast, macrophage depletion led to a very substantial decrease in the number of HSPCs/cd41:eGFP þ colonizing the CHT (Fig. 2g,h versus e,f and n; À 89%, n ¼ 28) and the thymus at 72 h.p.f. (Fig. 2l, versus k and n; À 91%, n ¼ 28), while injecting L-clodronate at 45 h.p.f. (Fig. 2i) caused less marked effects ( À 72% in CHT: Fig. 2j,n, n ¼ 28; and À 50% in the thymus: Fig. 2m,n, n ¼ 22). The partial colonization observed after late L-clodronate injections was likely due to HSPCs that had already left the AGM before macrophage depletion. It further proved that L-clodronate was not toxic for HSPCs (Fig. 2j).
To fully validate the effect of macrophage depletion on haematopoietic organ colonization, we set inducible nitroreductase genetic depletion of macrophages 26    experimental approach. Expression of E.coli nitroreductase under the control of mpeg1 promoter allows the inducible tissue specific and reversible depletion of macrophages by soaking embryos in the prodrug metronidazole. Similar to L-clodronate, metronidazole macrophage depleted embryos ( Fig. 3b versus a) exhibited an impaired CHT colonization by HSPCs/cd41:eGFP þ (Fig. 3b 0 versus a 0 , À 68% at 60 h.p.f., Fig. 3c, n ¼ 24) as well as an accumulation of HSPCs/cd41:eGFP in the AGM ( Supplementary  Fig. 3a, þ 71% at 60 h.p.f., n ¼ 11). Macrophages still present after 42 h of metronidazole treatment (Fig. 3b) result either from partial depletion or delayed depletion or both and most likely explain the partial CHT colonization by HSPC compared with embryos injected with L-clodronate (Fig. 3b 0 and Fig. 2g,h).
As the genetic depletion is reversible by removing metronidazole from the medium, we measured macrophage reappearance during the 2 days following prodrug withdrawal (Fig. 3d-g) and its effect on haematopoiesis in the CHT (Fig. 3d,e 0 ,f 0 ,g 0 ). Indeed the number of HSPCs/cd41:eGFP þ accumulated in the CHT was directly correlated with both the reappearance of macrophages in embryos ( Fig. 3d and Supplementary Fig. 3b) and the decrease of the HSPCs/ cd41:eGFP accumulation in the AGM (Supplementary Fig. 3a).
Altogether these results demonstrated the crucial role of macrophages in haematopoietic organ colonization.
Mmp-9 and mmp-13a are expressed by macrophages. To establish a direct connection between macrophages and mmps expression in the AGM, we combined L-plastin immunostaining (a leukocyte marker) with WISH of 48 h.p.f. embryos using mmp-2, mmp-9 and mmp-13a probes. Mmp-9 and -13a expression patterns ( Fig. 4g- As L-plastin is expressed by different myeloid populations, we analysed mmps expression in embryos injected with L-clodronate. We observed in the AGM that mmp-2 expression profile ( Supplementary Fig. 4g,g 0 ) was unchanged after macrophage depletion while mmp-13a (Figs 4l,l 0 ) was significantly reduced and mmp-9 completely disappeared (Fig. 4k,k 0 ).
We concluded that macrophages expressing mmp-9 are unambiguously associated with HSPC mobilization and intravasation.
Macrophage-mediated ECM degradation. WISH showed that mmp-9 is expressed by primitive macrophages, but no direct demonstration was made of macrophage-mediated matrix degradation. We therefore decided to visualize matrix degradation by in vivo zymography 30 using gelatin as an intermediate product of collagen degradation. We injected highly quenched fluorescein-labelled gelatin (Gelatin-FITC) into the muscles of Tg(mpeg1:mCherry) embryos (Fig. 5a, n ¼ 10). Degradation of this substrate releases FITC from its quencher resulting in green fluorescence. Specific punctiform green fluorescence was observed around spherical cells that appeared as unlabelled ghosts (Fig. 5b, that these migrating round cells are HSPCs, we injected Gelatin-FITC into Tg(kdrl:caax:mCherry) embryos in which vascular endothelium and HSPCs express the mCherry protein 10 . We observed the same specific punctiform fluorescence around HSPCs/mCherry þ cells emerging from the wall of the aorta (Fig. 5n-p, n ¼ 4).
In vivo zymography therefore provided a final demonstration of matrix degradation in the AGM by macrophage secreted proteases followed by HSPC mobilization and vein intravasation.

Discussion
The studies described herein show an unexpected accumulation of primitive macrophages in the AGM region (ventrally to the DA) of human and zebrafish embryo.
We previously reported that zebrafish HSPCs emerge in the subaortic mesenchyme then enter the PCV circulation through peculiar structures, which facilitate HSPC intravasation 9 . Live imaging in HSPC-cd41:eGFP þ /macrophage-mpeg1:mCherry þ zebrafish embryo shows these tiny entry points are created by macrophages during their migration through the stroma, which will allow HSPCs to join the PCV.
Macrophage depletion at early stages of development (25 h.p.f.) prevents the colonization of haematopoietic organs without affecting EHT in the AGM, while late depletion (45 h.p.f.) leads to less pronounced effects. Because zebrafish definitive haematopoiesis occurs between 30 and 55 h.p.f. (ref. 3), the partial colonization observed after late depletion is likely due to HSPCs which had already left the AGM before macrophage depletion. Therefore, our data suggest that primitive macrophages play an essential role in definitive haematopoiesis by inducing Mmpsmediated degradation of the ECM around HSPC in the AGM region. Matrix degradation allows mobilization and migration of HSPC into the stroma, driving their intravasation into the vein, leading ultimately to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis (as summarized in Fig. 6).
Another intriguing observation of this report is that a specific accumulation of primitive macrophages is also detected in the human embryo. CD68 þ macrophages are indeed concentrated in the mesenchyme beneath the ventral wall of the human embryonic aorta but also associated with the intra-aortic HSPC clusters. Even if, in the human, like any mammalian embryo the mobilization of HSPCs occurs within the DA circulation 1 , this accumulation of macrophages observed in the AGM, suggests that they could play a role in the establishment of definitive haematopoiesis also in these species. Therefore, we speculate that in human embryo, macrophages can facilitate the release of HSPCs from intra-aortic clusters, through a process evolutionary conserved.
Recent findings have demonstrated that macrophages maintain HSCs in adult bone marrow niches and their depletion mobilizes HSPCs in the bloodstream 31,32 . Our data show that during development, macrophages may have the opposite function by mobilizing HSPCs from the AGM followed by their intravasation and colonization of haematopoietic organs. This mobilization involves ECM degradation by the local secretion of Mmps.
Recent studies have shown that tumour-associated macrophages play a role in ECM remodelling in tumour stroma through the release of proteases from invadopodia, (mainly MMP-2 and 9) (ref. 18,19). These enzymes degrade the rigid stroma of tumours and enable CICs (cancer-initiating cells) to intravasate and seed secondary sites, that is, metastasis 33 . Our observations show marked similarities with this process, suggesting that HSPC and CIC migration might be comparable. It will thus be interesting to explore whether HSPCs also release factors that attract macrophages and what are their nature.

Methods
Human tissues and immunostaining. Human embryos were obtained from voluntary abortions performed according to guidelines and with the approval of the French National Ethics Committee. Patients gave their written consent for the embryos to be used for research purposes. Embryo sections (5 mm) were incubated with anti-CD34 (3.2 mg ml À 1 , Miltenyi) or anti-CD68 (10 mg ml À 1 , eBioscience) antibodies, then with biotinylated secondary antibodies (10 mg ml À 1 , Immunotech) and with peroxidase-labelled streptavidin (0.6 mg ml À 1 , Immunotech). Fluorescence-conjugated secondary antibodies (0.5 mg ml À 1 , Biolegend) were used for double staining.
Generation of mpeg1:mCherry-f transgenic Zebrafish line. A 1.9-kb mpeg1 promoter fragment encompassing the mpeg sequences driving promoter activity specifically in macrophages 38 was amplified by PCR using following forward 5 0 -TTGGAGCACATCTGAC-3 0 and NotI-introducing reverse 5 0 -TTATAGCGG CCGCGAAATGCTCTTGACTTCATGA-3 0 primers. Resulting product was digested by NotI and ligated to farnesylated mCherry protein coding sequence so that Mpeg1 ATG initiation codon would be in frame with downstream mCherry-F cds in a Tol2 derived vector. The resulting plasmid was injected along with transposase mRNA in one cell stage zebrafish embryos to achieve germline integration.
Macrophage chemical and inducible genetic depletion. Macrophages were depleted by injecting 5 nl of L-clodronate into the caudal vein (gift from M. Daeron Laboratory). L-PBS was injected as a control. Embryos with L-clodronate aggregates causing vessel occlusions and shunts were discarded.