Neuropilin 1 (NRP1) conveys SEMA3A signals to restrict physiological angiogenesis

doi:10.21203/rs.3.rs-6584058/v1

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Neuropilin 1 (NRP1) conveys SEMA3A signals to restrict physiological angiogenesis

https://doi.org/10.21203/rs.3.rs-6584058/v1

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The class 3 semaphorin SEMA3A is a secreted glycoprotein that serves as an evolutionary conserved axon repellent with proposed vascular functions. In mice, SEMA3A promotes vascular permeability in adults, but is dispensable for developmental brain, limb or trunk blood vessel patterning. By contrast, Sema3a restricts vessel branching in zebrafish embryo trunks. Whereas neuropilin 1 (NRP1) is thought to be the SEMA3A receptor in the mouse, prior reports identified Plxnd1 as the Sema3a receptor for zebrafish trunk vessel patterning, with no reported role for the zebrafish NRP1 orthologues, Nrp1a and Nrp1b, in this process. However, knockdown and knockout studies have yielded contradictory results on Nrp1 requirement for vessel patterning in zebrafish. To resolve conflicting prior information, we have refined the prior knockdown strategy to limit off target effects and generated mutant zebrafish embryos lacking both Nrp1a and Nrp1b to show that Nrp1 restricts trunk vessel patterning in a Sema3a-dependent manner. Moreover, we show that Nrp1 and Sema3a action does not involve the splicing regulation of Flt1, previously proposed to act downstream of Plxnd1, to restrict pro-angiogenic signals from the vascular endothelial growth factor VEGFA. In agreement, NRP1 is required in human endothelial cells for SEMA3A-induced repulsion. Together, these findings demonstrate that NRP1 mediates repulsive SEMA3A cues in endothelial cells to shape physiological vascular morphogenesis, in analogy to its role in axon guidance.

Neuropilin 1

SEMA3A

angiogenesis

zebrafish

HUVEC

Blood vessels distribute oxygen, nutrients and immune cells throughout the vertebrate body, and are a prerequisite for life in all vertebrates. During embryogenesis, a process termed angiogenesis allows blood vessels to vascularise organs as they form. In healthy adults, angiogenesis can be reactivated following injury or during disease. In both contexts, the microvasculature integrates diverse angiogenic and anti-angiogenic signals, which are sensed by endothelial cells (ECs), whereby the pro-angiogenic vascular endothelial growth factor VEGFA induces the formation of angiogenic sprouts that are led by endothelial tip cells [1, 2].

The transmembrane protein neuropilin 1 (NRP1) localises to tip cell filopodia to regulate sprouting angiogenesis [3]. As NRP1’s modular extracellular domain allows interaction with both VEGFA but also the class 3 semaphorin axon guidance cue SEMA3A [4], delineating the precise angiogenic role of NRP1 in different contexts has been challenging. For example, VEGFA binding to NRP1 promotes mouse retinal angiogenesis during postnatal development but is dispensable for embryonic brain and trunk angiogenesis in the mouse [5, 6]. By contrast, exogenous SEMA3A is antiangiogenic in the chick chorioallantoic membrane assay [7] and in mouse tumour models [8, 9]. Although endothelial SEMA3A deletion was reported to impair EC tip filopodia formation in the mouse postnatal retina in an autocrine manner, this did not cause obvious vascularisation defects [10]. Moreover, endogenous SEMA3A is dispensable to regulate angiogenesis in the mouse embryonic brain, limb and trunk, and lack of semaphorin signalling via NRP1 does not affect brain developmental angiogenesis [11, 12]. Nevertheless, Sema3a was reported to prevent ectopic trunk vessel sprouting in the zebrafish embryo [13]. Zebrafish NRP1 orthologues, termed Nrp1a and Nrp1b, have not been implicated in this process, which was proposed to be mediated by Plxnd1 [13] by upregulating the soluble form of the alternative Vegfa receptor Flt1 (sFlt1, also known as sVegfr1) [14]. In contrast, in the mouse, PLXND1 serves as a SEMA3E receptor to restrict trunk vessel sprouting independently of NRP1 [15, 16], In another reported difference between mouse and zebrafish, nrp1a null mutants generated by genome editing nucleases were reported to lack obvious vascular defects in the trunk [17], even though other studies showed that the morpholino-mediated knockdown of either nrp1a or both nrp1a and nrp1b genes reduces vessel sprouting in the zebrafish trunk [18–22].

To resolve contradictory evidence about the genetic requirement for Nrp1 in zebrafish angiogenesis, we have refined and improved the previously published morpholino-based knockdown method to exclude off target effects that non-specifically disrupt trunk development and generated double mutant zebrafish for both nrp1a and nrp1b. Moreover, we complemented genetic interaction studies in zebrafish with functional assays in human cells to demonstrate that NRP1 conveys SEMA3A signals during vascular morphogenesis.

Nrp1a and Nrp1b knockdown causes ectopic ISV sprouting in the zebrafish embryo trunk

We and others showed that a nrp1a translation-blocking morpholino (MO) that partially targets nrp1b (nrp1a(/b)-MO) [22] interrupted the extension of intersomitic vessels (ISVs) in the zebrafish embryo trunk [18, 22, 23]. As the MO dose used was associated with toxicity [22], we sought to re-investigate the role of Nrp1a and Nrp1b using refined approaches with reduced confounding off target effects. First, we generated chimeric zebrafish embryos with mosaic targeting of nrp1a and nrp1b to prevent embryo-wide MO toxicity from unspecifically affecting ISV sprouting. Thus, we injected Tg(fli1a:EGFP) embryos at 1-4 cell stage with the previously used dose of nrp1a(/b)-MO (0.6 pmol/embryo) versus mock controls, termed standard (Std)-MO, and then transplanted cells from these embryos into Tg(kdrl:mCherry) blastula-stage embryos. By contrast to prior knockdown studies that targeted nrp1a/b throughout the embryo, nrp1a(/b) knockdown ECs (EGFP+) in chimeric embryos were able to form ISVs that reached the dorsal side of the trunk at 36 hours post fertilisation (hpf), similar to ISVs composed of host ECs (mCherry+) (Fig. S1). Moreover, nrp1a(/b) knockdown ECs located within ISVs extended ectopic sprouts towards the somites (Fig. S1), which are maintained blood vessel free at this developmental stage, suggesting that they had lost sensitivity to a repellent cue.

Next, we refined the MO knockdown regimen by separately titrating nrp1a(/b)- and nrp1b-MOs for a toxicity study, in which we evaluated overall embryo morphology and scored for the presence of macroscopic anatomical defects and cardiac oedema in 2 days post fertilisation (dpf) Tg(kdrl:EGFP) embryos. Injecting nrp1a(/b)- and nrp1b-MOs at doses equal or below 0.025 pmol/embryo and 0.9 pmol/embryo, respectively, did not cause obvious toxicity (Fig. S2a,b). Then, we identified non-toxic doses for both MOs that did not alter ISV morphogenesis when injected singularly (subcritical doses) and co-injected them into 2 dpf Tg(kdrl:EGFP) embryos (Fig. 1a, all tested combinations are shown in Fig. S2c,d). We found that a combination of 0.01 pmol/embryo nrp1a(/b)-MO and 0.9 pmol/embryo nrp1b-MO efficiently reduced both Nrp1a and Nrp1b protein levels and caused the formation of ISV sprouts that ectopically crossed the somite region with high penetrance (Fig. 1a-c, Fig. S2c,d). The ectopic ISVs anastomosed in rostrocaudal direction with adjacent ISVs along the inner medial border of the somites without penetrating them, thereby forming bridge-like structures along the trunk and the tail. In summary, the double Nrp1a and Nrp1b knockdown prevented ISV repulsion from the somite region, agreeing with the observations made with chimeric embryos.

Genetic Nrp1a and Nrp1b targeting causes ectopic ISV sprouting in the zebrafish embryo trunk

In a third approach, we generated double mutant zebrafish embryos lacking both Nrp1 paralogues by combining the nrp1a^sa1485[24, 25] and nrp1b^fh278[26] mutant lines, which have premature termination codons within the Nrp1 a2 and a1 domains, respectively (Fig. 1d). Western blot analysis using an antibody specific for the Nrp1 C-terminal domain confirmed lack of full length Nrp1a and Nrp1b proteins in 5 dpf double mutants (Fig. 1e). When generated from a double heterozygous incross, double mutants were present at a slightly lower frequency than expected (Fig. 1f), suggesting that loss of both Nrp1a and Nrp1b causes a small fitness reduction. Nevertheless, surviving double mutants reached adulthood and were fertile. Similar to MO-mediated knockdown, the simultaneous loss of Nrp1a and Nrp1b in the Tg(kdrl:mCherry) background resulted in the formation of ectopic ISV sprouts at 2 dpf (Fig. 1g,h) with complete penetrance (Fig. 1i).

ISVs are first formed by primary sprouting from the dorsal aorta (DA) between 22-36 hpf followed by secondary sprouting from the posterior cardinal vein (PCV) at 32-48 hpf. Therefore, we evaluated if ectopic ISV sprouts are caused by defects in primary ISV sprouting. At 26 hpf, primary ISVs in both double nrp1a and nrp1b morphants and mutants elongated towards the dorsal side of the trunk, as seen in controls; however, in contrast to controls, they extended filopodia-studded vessel sprouts across the somite region in both central and caudal trunk regions (Figs. 2a-c, S3a,b, Movies M1, 2). Using the endothelial nuclear reporter line Tg(fli1a:nEGFP) alongside Tg(kdrl:mCherry), we found that ISVs in double morphants and double mutants were composed of a significantly higher number of ECs (Figs. 2a-c, S3a-c). This increased EC number was already observed before ectopic sprouts elongated towards the somite region and in newly generated sprouts that had not yet reached the dorsal trunk (Figs. 2a-c, S3a-c). As ECs positive for the mitotic marker phosphorylated histone H3 (pHH3) were too rare in a single time snapshot at 26 hpf for quantitative scoring (Fig. S3a-c), we instead investigated EC proliferation by scoring EC mitotic events in each ISV via time lapse analysis (Fig. 2d,e, Movie M3). Compared to controls, double mutants showed a significantly increased number of EC mitotic events (Fig. 2d,e, Movie M4). Increased proliferation was associated with a decrease in EC recruitment from either the dorsal aorta or posterior cardinal vein but, by 40 hpf, double mutant ISVs still contained a higher number of ECs than wild type ISVs (Fig. 2f). These findings suggest that lack of ISV repulsion from the somite region in double morphant or mutant embryos result in ISV overgrowth and ectopic sprouting that are fed by increased EC proliferation within each primary ISV sprout.

Sema3a expression during ISV sprouting in the zebrafish embryo trunk

The above complementary loss-of-function strategies all showed that the combined loss of Nrp1a and Nrp1b causes ISV expansion and ectopic sprouting across the somites, similar to the previously described phenotype caused by loss of Sema3a paralogue Sema3ab [13]. Moreover, both Sema3a paralogue genes, sema3aa and sema3ab, have been shown to be expressed in the somites between 15 and 24 hpf [13, 27-29]. To better understand the distribution of both Sema3a paralogues concomitantly to ISV morphogenesis, we performed whole mount in situ hybridisation for sema3aa and sema3ab between 24 and 48 hpf, when Nrp1 loss of function leads to ectopic ISVs. Both paralogues were expressed in the dorsal and ventral halves of the somites, with the sema3aa signal appearing more diffused and becoming barely detectable at 48 hpf, whereas the sema3ab signal showed a somite-specific pattern throughout the entire mediolateral extension of the somites at all stages examined (Fig. 3a-d, S4a). Both paralogues appeared to be significantly more abundantly expressed in the ventral than dorsal portion of the somites (Fig. 3a-d). These observations are consistent with Sema3aa and Sema3ab providing the chemorepulsive cues that prevent ISVs from sprouting across the somites, with Sema3ab, whose knockdown caused ectopic ISV sprouting (Fig. S4b), as previously reported [13], showing a more defined expression pattern that persisted in the somites throughout the ISV morphogenesis window.

Nrp1 and Sema3a cooperate to prevent ectopic ISV sprouting independently of sFlt1

To evaluate whether Nrp1 and Sema3a genetically interact, we therefore focussed on Sema3ab. We first determined subcritical doses of nrp1a(/b)-, nrp1b- and sema3ab-MOs that did not cause ectopic ISVs or other defects when injected. A combination of 0.01 pmol/embryo of nrp1a(/b)-MO with 0.1 pmol/embryo of nrp1b-MO and a single dose of 0.3 pmol/embryo of sema3ab-MO were found to meet these criteria (Fig. 3e,f, S2c,d). The co-injection of these subcritical doses of nrp1a(/b)-, nrp1b- and sema3ab-MOs, however, caused a significant number of ectopic ISV sprouts with 72% penetrance (Fig. 3e,f). Nrp1, therefore, cooperates with Sema3a to prevent vascular overgrowth in the zebrafish embryo trunk.

Sema3ab was previously hypothesised to restrict vascular sprouting in the zebrafish embryo trunk by signalling via Plxnd1 [13], which then inhibits vascular expansion by promoting the expression of the VEGFA trap sFlt1 downstream of alternative splicing of the flt1 gene [14]. Therefore, we measured transcript levels of the flt1 membrane (mflt1) and soluble (sflt1) alternative splicing isoforms in the trunk of 28 hpf embryos injected with either the combined doses of nrp1a(/b)-MO (0.01 pmol/embryo) and nrp1b-MO (0.9 pmol/embryo) or with the single dose of sema3ab-MO (0.6 pmol/ embryo) that induced ISV defects. However, RT-qPCR analysis detected a slight increase in the transcripts for either membrane-bound Flt1 (mflt1) or soluble Flt1 (sflt1) rather than a decrease, in morphants compared to controls (Fig. S4c,d). This increase was similar to that for transcripts encoding for the pan endothelial marker genes cdh5 and kdrl (Fig. S4c,d), which likely reflected the increased EC number in morphants (see Fig. 2). Expression analyses therefore suggest that Nrp1 mediates Sema3a-chemorepulsive signals during zebrafish vascular morphogenesis in the trunk without affecting sFlt1 expression.

NRP1 mediates SEMA3A repulsive cues in human ECs

To understand the cellular and molecular mechanisms by which SEMA3A and NRP1 cooperate to shape vascular morphogenesis, we co-cultured SEMA3A-expressing human embryonic kidney (HEK) 293T cells with human umbilical vein endothelial cells (HUVECs) (Fig. 4a). When intermixed with mock transfected HEK cells, HUVECs formed a dense monolayer, whereas they were significantly repelled by SEMA3A-expressing HEK 293T cells (Fig. 4b,c). Knockdown of NRP1 in HUVECs (Fig. 4d) via a previously validated siRNA [30] suppressed SEMA3A ability to repel ECs (Fig. 4b,c). These experiments demonstrate that NRP1 mediates SEMA3A chemorepulsive cues cell autonomously in ECs.

Our results suggest that the main role of Nrp1 during zebrafish trunk vascularisation is to mediate signals that restrict lateral blood vessel sprouting. These findings are based on the observations that double knockdown or knockout of Nrp1 zebrafish orthologues resulted in lack of ISV repulsion from the somite region (Fig. 1) and consequent ISV overgrowth (Fig. 2). This observation contrasts past studies using either a knockdown or a knockout strategy in zebrafish. In particular, prior studies using a MO-mediated knockdown of either Nrp1a or both Nrp1a and Nrp1b reported defective ISV extension towards the dorsal larval trunk [18–22]. As we had found that the dose most often employed in previous studies for the translation-blocking MO targeting both nrp1a and nrp1b was associated with general toxicity that prevented proper embryo development [22], we have here refined the Nrp1 knockdown approach to avoid off target effects: chimeric embryos with mosaic knockdown of nrp1a and nrp1b (Fig. S1) and combining subcritical doses of 2 different MOs (Fig. 1, S2). In both cases, ISV elongation towards the dorsal side of the trunk was not affected, whereas we observed ectopic ISV extension across the somite region (Fig. 1, S1, S2). Nrp1 knockout strategies in zebrafish have to date been limited to Nrp1a, without reported vascular defects [17], except slightly impaired collective EC migration in the common cardinal vein [31]. Consistent with these prior studies and with both Nrp1 paralogues being expressed in the ISVs with a similar spatiotemporal pattern [18, 19, 25], we found that both Nrp1a and Nrp1b were each individually dispensable for ISV formation. However, consistent with the refined double MO knockdown strategy, the simultaneous loss of both paralogues in double mutants resulted in ISVs with normal dorsal extension but ectopic invasion of the somite region (Fig. 1). Transcriptional adaptation is a recently described genetic compensation by which related gene(s) are upregulated downstream of mutant mRNA degradation [32], The redundant requirement for each single Nrp1 paralogue is unlikely due to transcriptional adaptation, because the nrp1a mutation employed in our study did not increase Nrp1b expression (Fig. 1)[25].

The only reported vascular defects for Nrp1a loss was slightly impaired Sema3d-induced collective EC migration in the common cardinal vein [31]. Consistent with an additional role for Nrp1 in mediating semaphorin signalling also in trunk ISVs, we have observed ectopic sprouting in both Nrp1a and Nrp1b double mutants and morphants at a developmental stage when the trunk region expresses the SEMA3A orthologues Sema3aa and Sema3ab (Fig. 3). In agreement with a role for NRP1 as a SEMA3A receptor, genetic interaction experiments in zebrafish showed that Nrp1a and Nrp1b prevent ectopic ISV sprouting in the somite region by cooperating with Sema3ab (Fig. 3), the Sema3a paralogue previously implicated with the modulation of vascular repulsion in zebrafish embryos [13]. Ectopic ISVs observed with the sema3ab-MO critical dose or with the triple combination of nrp1a(/b)-, nrp1b- and sema3ab-MOs subcritical doses were most frequent across the dorsal portion of the somite (Fig. 3), which may be explained by the most effective loss of Sema3ab in knockdown experiments in those regions that are less abundant in sema3ab transcripts compared to the ventral half of the somites (Fig. 3). SEMA3A has also been reported to inhibit EC proliferation during mouse kidney development [33] and in cultured human ECs [34]. In agreement, ectopic sprouting in Nrp1a and Nrp1b double mutants was accompanied by increased EC proliferation within each ISV (Fig. 2).

Rather than a predominant pro-angiogenic effect, as observed in the brain and retina of mice [3, 5, 22, 30, 35, 36], the main role of Nrp1 during trunk vascularisation in the zebrafish is to mediate signals that restrict blood vessel sprouting (Fig. 1, 3). Interestingly, this finding differs from findings in mouse embryos, in which SEMA3A is dispensable for trunk vascular patterning [11]. The discrepancy might be due to a more restricted and superficial SEMA3A expression in mouse embryonic somites [37] than in zebrafish, whereby sema3ab transcripts accumulated throughout the mediolateral extension of the somites (Fig. S4). SEMA3A or semaphorin signalling via NRP1 were also shown to be dispensable for vascularisation of the mouse embryonic hindbrain [11], where SEMA3A is expressed at low levels [38] when compared to the strong and highly stereotyped expression of SEMA3A orthologues in the zebrafish trunk (Fig. 3, S4). Importantly, in support of our zebrafish observations, we found that SEMA3A repelled human ECs via NRP1 (Fig. 4), in accordance with a previous report demonstrating that SEMA3A reduced ECs migration towards extracellular matrix cues in a NRP1-dependent fashion [39]. Moreover, our human cell assay showed that expression of NRP1 specifically in ECs mediates SEMA3A repulsive signals (Fig. 4). Such EC autonomous role for NRP1 in negatively regulating angiogenesis has been previously hypothesised to be partly complemented in pathological settings by an effect of SEMA3A on recruitment of NRP1-expressing monocytes [40].

Sema3a was previously suggested to promote ISV repulsion in zebrafish by binding to Plxnd1, which in turn induces upregulation of sFlt1 [14]. However, we found that mRNA transcripts for sFlt1 were not reduced by Sema3ab or Nrp1 loss of function (Fig. S4). Our results therefore indicate that NRP1, once bound by SEMA3A ligands, might engage in a complex with a plexin family member different from PLXND1. For example, PLXNA1 was previously shown to mediate SEMA3A inhibition of human EC migration towards extracellular matrix [39] and SEMA3A signals in lymphatic ECs for lymphatic valve morphogenesis [41]. Moreover, we recently demonstrated that PLXNA2 is the most abundantly expressed class A plexin in both human and mouse ECs [42]. Even though it is still possible that PLXND1 activation regulates sFLT1 expression, our data agree with lack of defects in ISV primary (and secondary) sprouting in zebrafish embryos lacking Flt1 [43, 44], further supporting that the EC repulsion induced by SEMA3A-NRP1 interaction is independent from the release of sFLT1.

In addition to class 3 semaphorins, NRP1 modular extracellular domain allows interaction with other ligands, such as VEGFA, with Vegfa signalling in zebrafish being essential to promote the sprouting and elongation of ISVs [45–47]. However, Nrp1 loss in our refined knockdown and knockout strategies did not reduce ISV elongation towards the dorsal side of the trunk (Fig. 1). Even though different studies reported NRP1 as a positive regulator of blood vessel morphogenesis in mouse [3, 12, 22, 30], a limited role for Nrp1 in Vegfa signalling in zebrafish still agrees with previous observations made in mouse embryos, whereby mutants lacking VEGFA binding to NRP1 do not show major vascular defects [5, 6].

In conclusion, our results resolve previous conflicting reports on the genetic requirement for Nrp1 in zebrafish angiogenesis by demonstrating a fundamental role for NRP1 in mediating endogenous SEMA3A repulsion cues for ECs during physiological vascular morphogenesis in vivo, a function that is also conserved in humans.

Author Contribution

M.S., C.R. and A.F. contributed to the conception and design of the study. M.S., C.R. and A.F. co-wrote the manuscript. M.S., E.G., F.F., V.C., G.G., S.P., M.T., L.D., C.P. and A.F. performed zebrafish experiments. M.S. and C.T. performed cell experiments. M.S., E.G., F.F. and A.F. analyzed data. All authors read and approved the submitted manuscript.

Acknowledgement

We thank the Animal Care unit and the NOLIMITS Unitech imaging facility at University of Milan, Jonathan A Raper for providing the nrp1 mutant zebrafish strain, Monica Beltrame, Mariya Moosajee and Dhani Tracey-White for technical assistances. This study was supported by research grants from the Fondazione Cariplo (2018-0298) and the Fondazione Associazione Italiana per la Ricerca sul Cancro (AIRC) (22905) to AF, British Heart Foundation (PG/18/85/34127) to AF and CR, Academy of Medical Sciences’ Springboard grant (SBF008\1139) to CP and Medical Research Council (MR/T020164/1) to GG. The funders had no role in the study design, data collection and interpretation, nor the decision to submit the work for publication.

Data Availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request.

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Neuropilin 1 (NRP1) conveys SEMA3A signals to restrict physiological angiogenesis

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