Results and Discussion
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 [7]. Hence, specification
of TCs involves tight spatiotemporal control of Vegfr/Notch signaling [8]. 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 [9], 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
[11]) 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 [12].
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
[4] (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 [13], 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
[18]. 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 [13], 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 (clo
s5
) mutants that lack endothelial tissues [19], 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 [4]. 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 [21])
of the DA, which express high levels of efnb2a (Figure 4D) [22]. 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 [18]. 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 [27], 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 [1], our
findings raise the exciting possibility that analogous mechanisms may also control
SC identity in other systems.