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Tissue branching morphogenesis requires the hierarchical organization of sprouting cells into leading “tip” and trailing “stalk” cells [1, 2]. During new blood vessel branching (angiogenesis), endothelial tip cells (TCs) lead sprouting vessels, extend filopodia, and migrate in response to gradients of the secreted ligand, vascular endothelial growth factor (Vegf) . In contrast, adjacent stalk cells (SCs) trail TCs, generate the trunk of new vessels, and critically maintain connectivity with parental vessels. Here, we establish that h2.0-like homeobox-1 (Hlx1) determines SC potential, which is critical for angiogenesis during zebrafish development. By combining a novel pharmacological strategy for the manipulation of angiogenic cell behavior in vivo with transcriptomic analyses of sprouting cells, we identify the uniquely sprouting-associated gene, hlx1. Expression of hlx1 is almost entirely restricted to sprouting endothelial cells and is excluded from adjacent nonangiogenic cells. Furthermore, Hlx1 knockdown reveals its essential role in angiogenesis. Importantly, mosaic analyses uncover a cell-autonomous role for Hlx1 in the maintenance of SC identity in sprouting vessels. Hence, Hlx1-mediated maintenance of SC potential regulates angiogenesis, a finding that may have novel implications for sprouting morphogenesis of other tissues.
► Expression of hlx1 is associated with angiogenic cell behavior in vivo ► hlx1 selectively marks sprouting endothelial cells during zebrafish development ► Hlx1 is required for intersegmental vessel angiogenesis in zebrafish embryos ► Hlx1 cell-autonomously maintains endothelial stalk cell potential
To identify previously unknown determinants of endothelial cell (EC) sprouting, we defined and exploited a pharmacological strategy for the manipulation of angiogenic cell behavior in vivo. Whereas high vascular endothelial growth factor receptor (Vegfr) signaling is known to promote tip cell (TC) specification, activation of the Notch receptor via its ligand Delta-like 4 (Dll4) represses the TC phenotype to promote stalk cell (SC) fate [4–6]. Conversely, suppression of Notch activity upon antagonistic interaction with its ligand Jagged1 promotes TC formation . Hence, specification of TCs involves tight spatiotemporal control of Vegfr/Notch signaling . Consequently, we hypothesized that the pharmacological manipulation of Vegfr/Notch signaling selectively during zebrafish intersegmental vessel (ISV) angiogenesis would enable the precise control of angiogenic EC behavior and sprouting-associated gene expression in vivo. In control dimethyl sulfoxide (DMSO)-treated Tg(kdrl:GFP)s843 embryos , green fluorescent protein (GFP)-expressing ECs sprout by angiogenesis at regular intervals from the first embryonic blood vessel, the dorsal aorta (DA), to form the ISVs. Nascent ISVs then connected with adjacent ISVs to form the dorsal longitudinal anastomotic vessel (DLAV) at 30 hr postfertilization (30 hpf; Figure 1A) [4, 10]. Quantification of EC numbers in sprouting ISVs using a nuclear-localized endothelial-specific enhanced green fluorescent protein (EGFP) transgene (Tg(kdrl:nlsEGFP)zf109 ) showed that ISVs at 30 hpf stereotypically contain three to four ECs, as previously reported [4, 11] (Figure 1B). However, using established pharmacological inhibitors of the Vegfr and Notch signaling pathways (SU5416 and DAPT, respectively), we were able to precisely manipulate sprouting EC numbers during ISV angiogenesis (Figures 1B–1D). EC sprouting was significantly enhanced upon incubation of embryos with DAPT from prior to ISV sprouting (22 hpf) to 30 hpf (Figures 1B and 1C), consistent with the EC hypersprouting phenotypes observed in the absence of Notch signaling [4–6]. In contrast, EC sprouting was entirely blocked in embryos incubated with high levels of Vegfr inhibitor (2.5 μM SU5416, Figures 1B and 1D), as previously observed . Moreover, serial dilution of SU5416 (see Figures S1A and S1B available online) revealed that intermediate EC-sprouting phenotypes could be obtained upon partial inhibition of Vegfr (0.625 μM SU5416; Figures 1B and 1D). Hence, temporal disruption of Vegfr/Notch signaling during ISV sprouting allowed precise pharmacological control of angiogenic versus nonangiogenic EC behavior in vivo.
Exploiting these observations, we defined a novel strategy to identify genes functionally associated with EC sprouting (Figure 1E). Tg(kdrl:GFP)s843; Tg(gata1:DsRed)sd2 embryos were incubated from 22 to 30 hpf with compounds that either promoted (DAPT), fully repressed (2.5 μM SU5415), or partially repressed (0.63 μM SU5416) angiogenic cell behavior in vivo (Figures 1A–1D; Figure S1). Pharmacologically manipulated GFP-positive ECs were then isolated by fluorescence-activated cell sorting (FACS) and separated from GFP/dsRed-double-positive erythrocytes prior to comparison of their transcriptomes to DMSO-treated controls. Subsequent multifactorial comparison of expression profiles (see Experimental Procedures) identified 109 genes whose expression was tightly correlated with EC-sprouting levels, including flt4, the only known TC-enriched gene in zebrafish  (Figure 1F; Figures 1C and S1D). Surprisingly, the most SU5415/Vegfr responsive of these genes was a homeobox transcription factor gene, h2.0-like homeobox-1 (hlx1) (Figure 1F; Figure S1C), which displayed an expression profile that was highly correlated with EC-sprouting levels (Figure 1F; Figure S1D). Furthermore, expression analyses revealed that compared to the pan-endothelial marker kdrl , hlx1 was highly enriched in sprouting ECs in vivo (Figure 1G), suggesting a key role for Hlx1 during ISV angiogenesis. The mammalian ortholog of Hlx1 (HLX) was originally identified as a key determinant of mammalian liver, gut, and hematopoietic development [14–17]. Strikingly, Hlx null mice also display features of severe vascular dysfunction (edema, early lethality) [16, 17], and HLX was recently shown to influence expression of EC guidance cues in vitro . However, the vascular function of HLX/Hlx1 in vivo is unknown.
To confirm an association of hlx1 with angiogenic cell behavior in vivo, we assessed its spatiotemporal pattern of expression during zebrafish development (Figures 2A–2J). Compared with expression of the EC marker kdrl , hlx1 expression was not detected in the first embryonic artery (DA), which forms by the process of vasculogenesis (red bracket in Figures 2A and 2B) [10, 12]. However, during ISV angiogenesis, hlx1 expression was enriched in the first-sprouting ECs (arrows in Figures 2C and 2D). Expression of hlx1 was also observed prior to ISV sprouting at discrete regions of future angiogenic remodeling within the DA (arrowheads in Figure 2D). At subsequent developmental stages hlx1 became increasingly enriched in sprouting ISVs (Figures 2E and 2F) and was almost exclusively restricted to angiogenic ECs at 30 hpf (Figures 2G and 2H). Similarly, sprouting angiogenic ECs of the midcerebral veins (MCeVs) were also hlx1 enriched (arrows in Figures 2I and 2J). However, hlx1 expression was excluded from the adjacent nonangiogenic parental tissues of the DA and primordial hindbrain channel (PHBC) during ISV and MCeV angiogenesis (Figures 2G–2J). Importantly, vascular expression of hlx1 was also absent in zebrafish cloche (clos5) mutants that lack endothelial tissues , confirming expression of hlx1 in sprouting ECs (Figures 2K and 2L). Furthermore, EC hlx1 expression was reduced or lost upon the SU5416-mediated disruption of ISV sprouting (Figures 2M and 2N). In contrast, DAPT-induced EC hypersprouting promoted ectopic hlx1 expression throughout the endothelium (arrowheads in Figure 2O), which could be blocked upon coincubation of embryos with SU5416 (Figure 2P). Finally, mature ISVs at stages after angiogenesis no longer expressed hlx1 (data not shown). Hence, expression of hlx1 exclusively marks sprouting ECs and represents a unique marker of angiogenic versus nonangiogenic ECs.
To elucidate the function of Hlx1 during ISV angiogenesis, we injected Tg(kdrl:GFP)s843 and Tg(kdrl:nlsEGFP)zf109 embryos with either control morpholino oligonucleotides (MOs) or hlx1-targeting MOs that disrupted hlx1 translation or exon-intron splicing (Figure S2). ISV sprouting in hlx1 MO-injected (morphant) embryos was delayed at 30 hpf and severely disrupted at 48 hpf (Figures 3A–3G). In particular, ISVs in hlx1 morphant embryos were predominantly stunted, failed to connect with adjacent vessels, and often remained blunt ended (asterisks in Figure 3A). Hlx1 knockdown did not notably affect embryo morphology (Figure 3H), arterial/venous differentiation of ECs (Figure S3A), assembly of the axial vessels (Figure 3I), or blood flow through the DA (red arrow in Figure 3J) and cardinal vein (blue arrow in Figure 3J). However, injection of embryos with hlx1 MOs severely disrupted blood flow through the ISVs (Figure 3J), consistent with inadequate angiogenesis and reduced connections between adjacent vessels (Figures 3A and 3I). Importantly, perturbed ISV sprouting was associated with decreased incorporation of ECs into sprouting vessels (Figures 3D–3F) and reduced EC proliferation (Figure 3G). Hence, consistent with its specific expression in sprouting ECs, hlx1 appears to be essential for ISV angiogenesis during zebrafish development.
Signaling via the Vegf-Notch axis promotes TC specification and behavior, at least in part, by inducing the TC-restricted expression of flt4 [4, 13, 20]. Consequently, flt4 morphant embryos display defects in EC sprouting similar to those observed upon Hlx1 knockdown . However, TC-associated expression of flt4 was comparable to controls in hlx1 morphant embryos, indicating that TC specification was unaffected (Figure S3A). Furthermore, observations that EC sprouting in hlx1 morphants was highly Flt4 dependent (Figures S3B and S3C) suggested that Hlx1-compromised ISVs still displayed TC behavior. Moreover, live-imaging analyses of Tg(kdrl:nlsEGFP)zf109 embryos revealed that the initial timing of TC migration was also unaffected in hlx1 morphant embryos (Movies S1 and S2). However, the hierarchical organization of sprouting cells was disrupted in hlx1 morphants versus controls (Movie S2). In particular, unlike in controls, sprouting ECs in hlx1 morphants did not rapidly sort into leading TCs and trailing SCs but appeared to display prolonged competition for the TC position (see ISVs B and C in Movie S2). Consequently, we hypothesized that Hlx1 may alternatively influence SC identity. Consistent with this hypothesis, we observed that hlx1 was uniquely expressed in the SC domain (as well as in TCs) during ISV sprouting (Figures 4A–4D). Whereas all vessels expressed the pan-endothelial marker kdrl (Figure 4A), hlx1 expression was restricted to angiogenic-sprouting cells of the ISVs (Figure 4B). However, unlike expression of flt4, which was restricted to the TC domain of sprouting ISVs (Figure 4C), hlx1 expression was expanded throughout the SC domain (Figure 4B). Moreover, hlx1 was excluded from adjacent nonangiogenic ECs (or “phalanx” cells ) of the DA, which express high levels of efnb2a (Figure 4D) . Other SC-enriched genes, such as flt1 and tie2, are also expressed at high levels in adjacent nonangiogenic tissues [23, 24]. Hence, to the best of our knowledge, hlx1 represents the first-identified discriminative marker of sprouting SCs versus nonangiogenic ECs.
SC-enriched expression suggested that hlx1 influences SC identity. To elucidate the cell-autonomous role of Hlx1 in SC fate decisions at single-cell resolution, we transplanted cells from Tg(kdrl:GFP)s843 embryos into nontransgenic hosts. Previous studies have determined that the TC and SC potential of ECs can be assessed during zebrafish development based on the ability of transplanted ECs to contribute to the DLAV or ISV stalk position, respectively [4, 25, 26]. Hlx1 alone was not sufficient to induce SC fate because hlx1-RNA-injected donors contributed to the DLAV and ISV stalk positions with a similar frequency as controls (Figures 4E and 4F; Table S1). However, unlike controls, cells transplanted from hlx1-MO-injected donors frequently contributed exclusively to the DLAV position of ISVs (Tip, Figure 4E). Importantly, quantification of the positional fates of donor cells within ISVs confirmed that Hlx1-knockdown ECs were less likely to acquire SC fate, preferentially migrated to the DLAV position, and were found exclusively at the DLAV position of at least 42% of sprouting ISVs (Figure 4F; Table S1). Furthermore, live-imaging studies revealed that cells transplanted from hlx1 morphant embryos, unlike controls, frequently exclusively occupied the leading TC position and did not contribute to ISV SCs (Movies S3 and S4). These data lead us to propose that Hlx1 functions cell-autonomously to reinforce and maintain SC potential during ISV sprouting (Figure 4G). Hence, Hlx1 regulates angiogenesis by influencing the outcome of EC competition for the TC position.
Previous work has primarily focused on defining the roles of VEGFR and Notch signaling in TC formation during new blood vessel sprouting [3–8]. Here, we show that Hlx1-mediated maintenance of SC potential appears to be critical for normal ISV angiogenesis in vivo (Figure 3). Whereas TCs express high levels of promigratory genes (such as vegfr2 and flt4) [4, 6, 20] and are highly motile, SCs need to be characteristically less motile to maintain their position behind TCs. HLX was recently found to impede the migratory behavior of ECs in vitro by inducing the expression of repulsive guidance molecules such as UNC5B . Hence, Hlx1-mediated repression of EC migration may be critical for determining SC positioning and functional blood vessel sprouting. In addition, our findings indicate that Hlx1 may also positively influence EC proliferation, because a decrease in cell divisions was observed in the ISVs of hlx1 morphant embryos (Figure 3G). Moreover, this proliferation defect may account, at least in part, for the reduced number of ECs in the sprouting ISVs of hlx1 morphants (Figures 3D–3F), which contrasts sharply with the hypersprouting phenotype observed in mosaic hlx1-deficient ECs (Figures 4E and 4F). Most importantly, our findings provide new evidence that reinforcement of SC identity and positional fate is critical for angiogenesis and implicate Hlx1 in this process. Hence, a fine balance between TC- and SC-inducing signals is crucial for the coordinated sprouting of new blood vessels. Considering the potential therapeutic implications of manipulating SC formation during pathological angiogenesis , future studies defining the downstream transcriptional network and precise cellular mechanisms of Hlx1 function in vivo will be of great importance. Furthermore, because the global mechanisms controlling angiogenesis and the branching morphogenesis of various epithelial tissues appear to be highly conserved , our findings raise the exciting possibility that analogous mechanisms may also control SC identity in other systems.
S.P.H. is a Wellcome Trust Research Career Development Fellow. This study was supported in part by grants from the National Institutes of Health (HL54737) and Packard Foundation to D.Y.R.S. Zebrafish studies were carried out in a designated facility under licenses issued by the United Kingdom Home Office.
Embryos were imaged from 19 hpf for approximately 13 hr. ISV sprouting is initiated by a single endothelial tip cell that is either trailed by an endothelial stalk cell (see ISV B) or divides to form two cells that rapidly hierarchically organize into leading tip and trailing stalk cells (see ISVs A and C).
Embryos were imaged from 19 hpf for approximately 13 hr. Multiple cells frequently sprout into newly forming ISVs and display defects in their hierarchically organization into leading tip and trailing stalk cells (see ISVs B and C).
Embryos were imaged from 20 hpf for approximately 16 hr. Donor cells are capable of contributing to both the tip and stalk cell position of sprouting ISVs.
Embryos were imaged from 20 hpf for approximately 16 hr. Donor cells frequently preferentially contribute to the tip cell position of sprouting ISVs.