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. Author manuscript; available in PMC: 2017 Mar 1.
Published in final edited form as: Dev Biol. 2016 Jan 6;411(1):115–127. doi: 10.1016/j.ydbio.2016.01.002

Vegfa signaling promotes zebrafish intestinal vasculature development through endothelial cell migration from the posterior cardinal vein

Andrew L Koenig 1, Kristina Baltrunaite 1, Neil I Bower 2, Andrea Rossi 3, Didier Y R Stainier 3, Benjamin M Hogan 2, Saulius Sumanas 1,✉
PMCID: PMC4769896  NIHMSID: NIHMS752496  PMID: 26769101

Abstract

The mechanisms underlying organ vascularization are not well understood. The zebrafish intestinal vasculature forms early, is easily imaged using transgenic lines and in-situ hybridization, and develops in a stereotypical pattern thus making it an excellent model for investigating mechanisms of organ specific vascularization. Here, we demonstrate that the sub-intestinal vein (SIV) and supra-intestinal artery (SIA) form by a novel mechanism from angioblasts that migrate out of the posterior cardinal vein and coalesce to form the intestinal vasculature in an anterior to posterior wave with the SIA forming after the SIV. We show that vascular endothelial growth factor aa (vegfaa) is expressed in the endoderm at the site where intestinal vessels form and therefore likely provides a guidance signal. Vegfa/Vegfr2 signaling is required for early intestinal vasculature development with mutation in vegfaa or loss of Vegfr2 homologs causing nearly complete inhibition of the formation of the intestinal vasculature. Vegfc and Vegfr3 function, however, are dispensable for intestinal vascularization. Interestingly, ubiquitous overexpression of Vegfc resulted in an overgrowth of the SIV, suggesting that Vegfc is sufficient to induce SIV development. These results argue that Vegfa signaling directs endothelial cells to migrate out of existing vasculature and coalesce to form the intestinal vessels. It is likely that a similar mechanism is utilized during vascularization of other organs.

Keywords: zebrafish, vegf, intestinal, vascular endothelial, organ

Introduction

Proper vascularization is necessary for development of the embryo and survival of the adult. A functional vascular network is required for the transport of nutrients, hormones, immune cells, and oxygen to cells, as well as the removal of potentially toxic metabolic waste products. The early development of major blood vessels, as well as the signals that promote vascular development, has been well characterized; however, little is known about organ-specific vascularization. Two distinct mechanisms have been implicated in vascular development. Vasculogenesis involves the differentiation of angioblasts from mesoderm and the formation of primitive blood vessels from angioblasts at the site of their origin, while angiogenesis refers to the formation of new vasculature from preexisting vessels by sprouting or vessel remodelling (Sabin, 1917). It has been suggested that organ specific vasculature develops by a combination of vasculogenesis and angiogenesis. For example, in the human liver, arteries are thought to develop by angiogenesis while highly fenestrated capillaries have been proposed to form by vasculogenesis from intra-hepatic mesenchyme (Gouysse et al., 2002). During mouse liver development, loose endothelial cells have been observed that intercede between the hepatic endoderm cells and the septum transversum mesenchyme (Kingsbury et al., 1956; Matsumoto et al., 2001; Sherer, 1991). However, the source of these endothelial progenitors has not been known and the genetic pathways involved in the different mechanisms of organ vascularization have not been studied in detail in any model system. Understanding the mechanism that leads to organ vascularization can give insight into the formation of vascular malformations and pathological angiogenesis.

The zebrafish has become an exceptional model for studying vascular development due to the transparency of the embryo and the ability for genetic manipulations (Bradbury, 2004). As in other vertebrate organisms, the cardiovascular system is among the first to form, with circulation beginning by approximately 24 hours post fertilization (hpf) (Fishman and Chien, 1997). The mechanisms and pathways involved in vascular development have also been shown to be highly conserved among vertebrates (Jin et al., 2005; Pardanaud et al., 1987). The axial vessels—the dorsal aorta (DA) and posterior cardinal vein (PCV)—develop during mid-somitogenesis from progenitors that originate bilaterally in the lateral plate mesoderm and migrate to the midline in two distinct waves, where they coalesce (Kohli et al., 2013; Torres-Vazquez et al., 2003; Zhong, 2005). Following formation of the axial vessels, intersomitic arteries sprout from the DA driven by Vegfa-Vegfr2 signaling (Bahary et al., 2007; Habeck et al., 2002; Siekmann and Lawson, 2007) and intersomitic veins from the PCV driven by Vegfc-Vegfr3 signaling (Hogan et al., 2009a), both at the somite boundaries.

Vascular endothelial growth factor (Vegf) signaling is required for development of blood vessels by directing the migration and survival of endothelial cells. This requirement of Vegf signaling for functional vascular development has been demonstrated in mouse (Carmeliet et al., 1996; Ferrara et al., 1996), Xenopus and chick (Cleaver and Krieg, 1998), as well as zebrafish (Lawson et al., 2002; Nasevicius et al., 2000). As in other vertebrates, zebrafish have multiple Vegf ligand and receptor pathways including Vegfa-Vegfr2 and Vegfc-Vegfr3. Zebrafish express two VEGFR2 homologs known as Kdrl/Flk1 and Kdr/Kdrb, and a single VEGFR3 homolog Vegfr3/Flt4 (Covassin et al., 2006). It has also been suggested that Kdrl could represent Vegfr4, a Vegf receptor that has been lost in mammals (Bussmann et al., 2008). Morpholino knockdown of vegfaa, one of the two mammalian Vegfa homologs in zebrafish, results in a loss of intersomitic vessels (ISVs) and caudal plexus, in addition to failure of axial vessel patterning, and lack of circulation (Childs et al., 2002; Nasevicius et al., 2000), while the combined knockdown of VEGFR2 homologs by injecting kdr morpholino (MO) into kdrl mutant embryos exhibits a milder yet similar phenotype (Covassin et al., 2006). Knockdown of Vegfc-Vegfr3 signaling results in loss of lymphatic vessels, as well as the inhibition of venous intersomitic vessel sprouting (Covassin et al., 2006; Karkkainen et al., 2004; Siekmann and Lawson, 2007).

The intestinal vasculature in zebrafish made up of the supra-intestinal artery (SIA) and sub-intestinal vein (SIV) are among the next vessels to develop in the trunk, following the axial vessels and ISVs. These vessels develop close to the surface in a highly stereotypic pattern and therefore are an excellent model to study organ specific vascularization. As analyzed by microangiography, the intestinal vasculature begins developing approximately 2.5 dpf and is complete by 4 dpf. The SIV begins to develop at 2.5 dpf as two bilateral vessels, of which the right vessel further develops, while the left vessel breaks up to form hepatic vasculature and loops to the right SIV by 4 dpf. The SIA develops around 3 dpf as an extension of the anterior mesenteric artery, and later forms connections with the SIV (Isogai et al., 2001). While the structure and formation of these vessels has been characterized, the mechanism driving the formation and the lineage of the endothelial cells that make up the vessels have not been identified.

Various studies have noted effects on the development of the SIV following genetic manipulation; however, an in detail examination has not been conducted. Vegf inhibition by chemical inhibitor and vegfaa morpholino results in a failure of SIV formation, and overexpression of vegfaa165 results in expansion of the SIV (Hao et al., 2010; Kawamura et al., 2008). In addition, overexpression of bmp2b or loss of microsomal triglyceride transfer protein results in ectopic sprouting from the ventral region of the SIV (Avraham-Davidi et al., 2012; Wiley et al., 2011). Additionally, MO knockdown of survivin or vegfab result in loss of SIV development (Bahary et al., 2007; Ma et al., 2007).

In this study, we demonstrate that the intestinal vasculature in zebrafish does not form by vasculogenesis de novo as it has been suggested by earlier studies in mammalian models, but rather that it forms via the migration of individual endothelial cells derived from the posterior cardinal vein which coalesce to form the sub-intestinal vein and the supra-intestinal artery. This is a novel mechanism, which differs from standard definitions of vasculogenesis and angiogenesis. We also examine the roles of multiple Vegf ligands and receptors and identify, through morphant and mutant analysis, Vegfaa-Vegfr2 signaling as essential for proper patterning of the intestinal vasculature. Interestingly, despite its role in venous ISV development, Vegfc-Vegfr3 signaling appears to be dispensable for the intestinal vasculature formation based on mutant analysis. However, we demonstrate that overexpression of vegfc results in ectopic ventral sprouts from the SIV, suggesting that Vegfc signaling is sufficient to induce SIV sprouting. Together these results demonstrate a working model of organ specific vascularization in which terminally differentiated endothelial cells migrate from existing vasculature to coalesce in forming new vessels. Because the molecular mechanisms in vascular development are highly evolutionarily conserved, it is likely that organ-specific vascularization proceeds similarly also in other vertebrates.

Materials and Methods

Zebrafish Lines and Embryos

The following zebrafish lines were used in experiments: Tg(fli1a:EGFP)y1 (Lawson and Weinstein, 2002), Tg(kdrl:mCherry)ci5 (Proulx et al., 2010), Tg(etv2:mCherry)zf373 (Veldman and Lin, 2012), Tg(fli1a:nEGFP)y7 (Roman et al., 2002), TgBAC(etv2:Kaede)ci6 (Kohli et al., 2013), Tg(tp1:eGFP)um14 (Parsons et al., 2009), vegfcum18 (Villefranc et al., 2013), Tg(hsp70:Vegfc)ci25 (Davis et al.), flt4sa9798 (Busch-Nentwich, 2013), flt4hu4602 (Hogan et al., 2009b), vegfaabn1, and kdrlum19 (Covassin et al., 2009). Embryos were incubated at 28.5°C and staged using criteria previously described (Kimmel et al., 1995). Embryos beyond 24 hours post fertilization (hpf) were treated with 1-phenyl-2-thiourea (PTU) to inhibit the formation of pigment. kdrl−/− embryos were identified at 24–48 hpf by failure of inter-segmental vessel extension and vegfaa−/− embryos were identified by failure to develop normal axial and intersomitic vasculature.

In situ hybridization

In situ hybridization was performed as previously described (Jowett, 1999). Anti-sense riboprobes for kdrl, fli1a, vegfc (Cermenati et al., 2013), kdr (Covassin et al., 2006), and flt4 labeled with DIG-UTP were synthesized using T7 RNA polymerase (Ambion/Promega) as previously described (Thompson et al., 1998). vegfaa probe was synthesized from PCR products derived from pCS2-vegfaa121 plasmid (Liang et al., 2001) using the primers below designed with an integrated T7 promoter site. Processed embryos were dehydrated in 100% ethanol for storage at −20°C to improve contrast then rehydrated in PBS and mounted in 3% methylcellulose under cover slips for imaging. An AxioImager compound microscope with Plan-Neofluar 10X/0.3 NA objective and AxioCam ICC3 color camera (Carl Zeiss Inc., USA) controlled using AxioVision 4.6 software (Carl Zeiss Inc., USA) was used to capture Z-stack images and produce extended focus images. Image contrast and brightness was adjusted using Adobe Photoshop CS6. Primer sequences: vegfaa probe fw:

  • 5’-TTATTTCTCGCGGCTCTCCTC-3’,

    vegfaa probe rev:

  • 5’-GAAATTAATACGACTCACTATAGGGCATCTTGGCTTTTCACATCTTTCT-3’.

Confocal and Time-Lapse Microscopy

All fluorescent images were obtained in live embryos mounted in 0.6% low melting point agarose with 0.004% Tricaine (and 0.003% PTU for time-lapse imaging) with a Nikon A1R upright confocal equipped with an apochromatic 16X/0.8 NA long working distance objective (Nikon Instruments Inc., USA). Images were obtained as z-stacks using Nikon IS Elements (Nikon Instruments Inc., USA). For time-lapse imaging, embryos were mounted in a 10 cm dish, covered with embryo medium containing 0.004% Tricaine and 0.003% PTU, kept on a heated stage at 32°C, and z-stacks were captured at 15 minute intervals for the duration of imaging. Maximum intensity projections were produced using either Nikon IS Elements or Fiji is Just ImageJ (Schindelin et al., 2012). Image contrast and brightness was adjusted using Adobe Photoshop CS6. All images are of right side sub-intestinal vein.

Vegf Receptor Inhibitor Chemical Treatment

SU5416/Semaxanib (Sigma, USA) was dissolved in DMSO to 42mM and stored at −20°C. Embryos were dechorionated and treated with 2.5μM SU5416 or DMSO (Sigma) in embryo medium beginning at 32 hpf and incubated at 28.5°C on a rocking platform. Embryos were imaged as described using an upright confocal microscope at 52–54 hpf.

Morpholino Microinjection

All morpholinos were obtained from GeneTools. Morpholino sequences are as follows: MO1-vegfaa translation blocking (5’-GTATCAAATAAACAACCAAGTTCAT-3’) (Nasevicius et al., 2000), MO1-kdr splice blocking (5’-GTTTTCTTGATCTCACCTGAACCCT-3’) (Covassin et al., 2006). Morpholinos were diluted to 2.5ng/nl in nuclease free water and injected into the yolk at the one cell stage and injected at 10 ng. Developmental delay of some morpholino-injected embryos was corrected for by allowing morphant embryos to develop until staging morphology matched wild type embryos.

Photoconversion of etv2:Kaede

Whole TgBAC(etv2:Kaede)ci6 embryos were photoconverted at 24 hpf using an AxioImager compound microscope with Plan-Neofluar 10X/0.3 NA objective. (Carl Zeiss Inc., USA). Embryos were placed in glass depression slides and exposed to DAPI-filtered (405 nm) light for 30 seconds each. Converted embryos were immediately screened to ensure complete photoconversion. Embryos were imaged as described using a confocal microscope.

Vegfc Overexpression

Tg(hsp70:Vegfc)ci25 line was generated by injecting tol2-hsp70:Vegfc; myl7:GFP-tol2 construct (Le Guen et al., 2014) together with tol2 mRNA. Transgenic embryos were identified by myl7:GFP expression and groups of transgenic and control embryos were raised at 28.5°C, heat shocked at 30 hpf for 1 hour at 37°C, and returned to 28.5°C for further raising. Embryos were screened for changes to SIV development and imaged at 52 hpf by confocal microscopy as described.

vegfcum18 and flt4sa9798 Genotyping

Groups of embryos of each line were collected, fixed and processed for in situ hybridization for fli1a. Following imaging as described, embryos were lysed and PCR was performed. PCR products for vegfcum18 were submitted for sequencing and mutant embryos identified by sequence results and flt4sa9798 were identified using the FspBI restriction enzyme (Thermo Scientific) to digest a site created by the mutation that is absent in wild type embryos. Sequence of genotyping primers were as follows:

  • flt4sa9798fw 5’-GAGTTTCTGTTGTTCCCATAGACTG-3’

  • flt4sa9798rev 5’-AAAGCATGAAACTCACTGGTTACG-3’

  • vegfcum18fw 5’-TCACTGAAAATATGACCTTTCTTCT-3’

  • vegfcum18rev 5’-TTGTGTGTTTGAACACTTACTCTGT-3’

Results

Intestinal vasculature is derived from the posterior cardinal vein

To characterize the formation of the intestinal vasculature we utilized a double transgenic line expressing Tg(kdrl:mCherry) (Proulx et al., 2010) and Tg(fli1a:eGFP) (Lawson and Weinstein, 2002). We performed time-lapse confocal imaging using this line beginning at 22 hpf – 24 hpf and observed the migration of individual fli1a:GFP+ endothelial cells out of the posterior cardinal vein (PCV) beginning at 28 hpf (Fig. 1A–C, Suppl. Fig. S1, Movies 1,2). These cells began migrating ventrally in an anterior to posterior wave and coalesced to form the primordial sub-intestinal vein (SIV) and later the supra-intestinal artery (SIA). Cells continued to migrate from the PCV forming the SIV through 56 hpf (Fig. 1C–G, Movies 1,2) and the SIA through 73 hpf (Fig. 1H–J, Movie 2). Multiple filopodial connections between the SIA and SIV were observed extending from the developing vessels (Fig. 2). Interconnecting vessels (ICVs) between the SIV and SIA formed only after angioblasts have coalesced into the primordial SIA and SIV vessels (Movies 3,4).

Figure 1.

Figure 1

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Cells from the posterior cardinal vein form the sub-intestinal vein, and the supra-intestinal artery. Confocal microscope timelapse images of live Tg(kdrl:mCherry);Tg(fli1a:EGFP) embryos beginning at approximately 25 hpf. (A,B) indicate region of interest displayed in other panels. Note that subintestinal vessel progenitors have very low kdrl:mCherry expression and appear green while endothelial cells within the axial vessels show both mCherry and GFP expression. (C) A thickened cell within the ventral wall of the PCV is observed which delaminates in (D) (arrows). Note the large heterogeneity of transgene expression within the wall of the PCV with mCherry negative cells interspersed next to mCherry positive cells. (E) Multiple SIV progenitors are observed (arrows) which still make contacts with the PCV. (F) SIV progenitors (arrows) acquire elongated shape with some of them still contacting the PCV. (G) SIV progenitors start to coalesce into a primordial vessel. Some still make contacts with the PCV (arrow). (H) SIV progenitors have largely coalesced into a vessel. Notice a new cell emerging from the PCV (arrow). (I) Multiple cells emerge from the PCV (arrows) and start coalescing into the SIA. Filopodia bridging the SIA and SIV are apparent. (J) Separate SIA and SIV primordial vessels are apparent. Anterior-left, dorsal-top.

Figure 2.

Figure 2

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Coalescence of angioblasts into the supra-intestinal artery and interconnecting vessel formation. Confocal microscope timelapse images of live Tg(kdrl:mCherry);Tg(fli1a:EGFP) embryos beginning at approximately 56 hpf. Note that subintestinal vessel progenitors have low mCherry expression and appear mostly green. (A,B) indicate region of interest displayed in other panels. (C–J) Maximum intensity projections of confocal images at selected time points. (C’–J’) Selected confocal sections were combined in a maximum intensity projection of the same region as in C–J to more clearly visualize SIA. At 56 hpf, portions of the SIA are already visible with few connections to the SIV (C,C’). Over a period of 24 hours (D– J’), cells from the PCV contribute to the further development of the SIA while branches from the SIV, indicated by arrowheads, extend toward the SIA and form interconnecting vessels (ICVs). Anterior-left, dorsal-top.

These results suggest that cells from the PCV give rise to both arterial SIA and venous SIV. To test if the SIA displays arterial identity, we examined Notch reporter tp1:GFP expression which is known to be expressed in the arterial vessels including the DA and excluded from venous vasculature including the PCV (Quillien et al., 2014). Tp1:GFP expression at 74 hpf was observed in the SIA but not SIV (Fig. 3) arguing that the SIA has arterial identity. Intriguingly, time lapse imaging using tp1:GFP; etv2:mCherry line demonstrated that initially SIA progenitors are largely tp1:GFP negative and they only acquire arterial identity during later stages of SIA formation, which is consistent with their venous origin (Suppl. Fig. S2, Movie 5).

Figure 3.

Figure 3

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SIA but not SIV displays arterial specific Notch reporter tp1:GFP expression. Confocal microscope images of Tg(etv2:mCherry);Tg(tp1:GFP) embryos. (A–C) GFP expression indicating Notch responsive cells is restricted to the DA, arterial ISVs, and the SIA at 74 hpf.

We next utilized the Tg(etv2:Kaede) line (Kohli et al., 2013) which expresses photoconvertible Kaede protein under a vascular endothelial specific etv2 promoter to further identify the origin of the progenitors for the SIV and the SIA. We confirmed that etv2:Kaede is localized to vascular endothelial cells as shown by co-localization with kdrl:mCherry (Suppl. Fig. S3) and then performed full embryo photoconversion at 24 hpf followed by imaging at 52 hpf (Fig. 4). All cells expressing the Kaede protein in the SIV and SIA demonstrated both red and green fluorescence (Fig. 4B–C”), indicating that they were present in the vasculature at the time of photoconversion, prior to initiation of SIV and SIA formation. Together, this demonstrates that the intestinal vasculature develops through migration of endothelial cells from the PCV, and not by angiogenic branching from existing vasculature or through de novo vasculogenesis.

Figure 4.

Figure 4

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Cells in the sub-intestinal vein are derived from existing vasculature. Confocal microscope images of live Tg(etv2:Kaede) embryos following photoconversion. Note that etv2:Kaede line exhibits mosaic expression and not all vascular endothelial cells are labeled. (A–A”) Unconverted control demonstrates that only converted cells are visible in the red channel. (B–B”) Photoconversion at 24 hpf prior to any formation of intestinal vasculature results in all Kaede-positive cells in the SIV being marked by the converted (red) form of Kaede. This argues that SIV progenitors are derived from existing endothelial cells already expressing etv2:Kaede in the embryo at 24 hpf. (C–C”) Cropped regions of converted embryos demonstrate that both the SIA and SIV are visible in the red channel. Arrows indicate SIV. Anterior-left, dorsal-top.

Vegf signaling is required for intestinal vessel formation

To determine if there is a Vegf signaling requirement for formation of the intestinal vessels, we used the pan-Vegf receptor inhibitor SU5416 (Fong et al., 1999; Serbedzija et al., 1999). Embryos were treated beginning at 32 or 44 hpf until imaging at 52hpf. SU5416 treatment at 32 hpf caused a regression and near complete failure of intestinal vascularization as well as partial regression of the inter-segmental vessels (ISVs) (Fig. 5). In contrast, treatment starting at 44 hpf did not significantly affect development of most embryonic vasculature including ISVs, but resulted in a severe inhibition of intestinal vasculature growth. This suggests that Vegf signaling has a specific role in intestinal vessel development.

Figure 5.

Figure 5

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Vegf signaling is necessary for intestinal vascularization. Confocal (A–C) and brightfield images (A’–C’) of live Tg(etv2:mCherry) embryos at 52 hpf. (B) 2.5μM SU5416 treatment from 32–52 hpf results in near complete loss of SIV as well as nearly complete regression of ISVs as compared to the DMSO treated control (A) , while milder defects are observed in both ISVs and SIV in embryos treated from 44–52 hpf (C). Arrows indicate fully formed (A) or fragmented SIV (B,C). Treated embryos display pericardial edema but otherwise are morphologically normal (A’–C’). Anterior-left, dorsal-top. Boxed region in brightfield images demonstrates area shown by confocal imaging.

Knockdown of vegfaa causes partial to full loss of intestinal vasculature

We then analyzed expression of the vegfaa homolog during intestinal vessel development. In addition to the previously reported vegfaa expression in the somites (Bahary et al., 2007), its expression at 48–72 hpf was observed in the endoderm along the yolk and yolk extension at the SIV and SIA formation sites (Fig. 6A–C’). This suggested that vegfaa may provide the guidance signal for angioblast migration and coalescence into the SIA and SIV. To test the functional requirement of vegfaa, we analyzed vegfaa mutants generated using TALEN (TAL effector nuclease) genome editing approach. An obtained vegfaa allele has a 10 bp deletion, resulting in a frame shift beginning after glutamine Q60 and a premature stop codon following serine S113, and is likely to represent a null allele. Similar to previously reported vegfaa MO results (Lawson et al., 2002; Nasevicius et al., 2000), vegfaa−/− embryos display greatly reduced or absent ISV development, have no blood circulation, develop pericardial edema and die at 4–7 dpf (Fig. 6E,F and data not shown). In addition to these defects, severe defects in SIV formation were apparent in vegfaa mutants. The SIV phenotype in these mutants at 54 hpf varied in severity from a moderate defect (partially assembled but greatly disorganized SIV, Class 1, 33/42) to nearly complete absence of the SIV (Class 2, 9/42) (Fig. 6D–F). The severity of the SIV defects correlated to the severity of ISV defects. The same embryos at 72 hpf (Fig. 6G–I) demonstrated that there is little recovery of more severe SIV defects. Quantification of endothelial cells in the SIV using nuclear-specific Tg(fli1a:nEGFP) (Roman et al., 2002) reporter line embryos demonstrate that vegfaa morphants have a reduced number of endothelial cells in the SIV at 54 hpf compared to wild type embryos (uninjected n=8: 97.3±3.5 cells, morphant n=23: 35.0±5.7 cells, p= 0.0002).

Figure 6.

Figure 6

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vegfaa provides a candidate guidance signal required for intestinal vessel development. in situ hybridization of wild type embryos (A–C’) and confocal images of live Tg(kdrl:mCherry) embryos (D–F’). (A–C) vegfaa is expressed at 28, 48, and 72hpf in a narrow stripe of cells within the endoderm along the yolk and yolk extension in the same region where angioblasts coalesce into the SIA and SIV. (A’–C’) show higher magnification of the embryos in A–C. (D–F) vegfaa−/− embryos at 54 hpf exhibit strong inhibition (classs 1) or complete absence of SIV development (class 2). In addition, single axial vessel and inhibition of intersegmental vessel angiogenesis, where ISVs are absent or do not extend past the midline, are observed. (D’–F’) The same embryos at 72 hpf show little to no recovery of more severe defects. (Class 1 – moderate SIV defect, Class 2 – absent or nearly absent SIV). Arrows indicate SIV. Anterior-left, dorsal-top.

Combined knockdown of zebrafish vegfr2 homologs causes defective patterning of intestinal vessels

We next analyzed the role of the primary receptors of Vegfa in zebrafish, Kdrl and Kdr, both homologs of human Vegfr2. We observed expression of kdrl and kdr in the developing SIV by in situ hybridization at 40–72 hpf (Fig. 7A–D). We therefore used the kdrlum19 (Covassin et al., 2009) mutant in addition to the previously validated kdr morpholino to analyze their role in intestinal vessel development (Covassin et al., 2006). We identified kdrl−/− mutants by failure of ISV extension and developed shunts at the midline as previously reported (Meng et al., 2008). In addition to the ISV phenotype, we also observed an underdeveloped and mispatterned SIV and SIA in kdrl−/− mutant embryos as compared to their siblings (Fig. 7E,F). kdr expression was not significantly affected in kdrl−/− embryos (Suppl. Fig. S4). kdr morphants displayed similar but milder phenotype in addition to a slightly bent body axis and mild pericardial edema (Fig. 7I,J), which included underdeveloped and mispatterned SIV and SIA (Fig. 7G). However, combination of kdr morpholino in the kdrlum19 background resulted in a more severe phenotype. ISVs were either absent or more severely stunted and axial vasculature was mispatterned similar to vegfaa−/− embryos, while SIV and SIA were completely absent (Fig. 7H). This establishes that both Kdr and Kdrl play essential and partially overlapping roles in the proper patterning of the zebrafish intestinal vasculature.

Figure 7.

Figure 7

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Vegfr2 homologs Kdr and Kdrl function redundantly in SIV development. (A–D) In situ hybridization analysis of kdrl and kdr expression in the SIV progenitors (arrows) in wild-type embryos at 48–72 hpf. (E–H) As analyzed by confocal imaging of live fli1a:GFP embryos, kdrl−/− mutants (F) display an underdeveloped and mildly mispatterned SIV that lacks connections to the SIA and form only partially extended intersegmental vessels, while kdr morphants (G) also demonstrate mispatterned and underdeveloped ISVs and partial SIV development, but retain connections to the SIA. Combined kdrl−/−, kdr morphant embryos (H) display more severe axial vessel, ISV, and SIV defects than the kdrl−/− alone, resembling vegfaa−/− embryos. (I,J) As shown by brightfield imaging of live embryos, kdr morphants display a slight curvature of the body axis (J), but are otherwise phenotypically normal compared to uninjected controls (I). Large arrows indicate SIV. Anterior-left, dorsal-top.

Vegfc/Flt4 signaling is dispensable for intestinal vascularization, but sufficient to induce ectopic growth

To test for a potential contribution of Vegfc-Flt4 signaling pathway to intestinal vessel development, we first analyzed expression of the vegfc and flt4 homologs during intestinal vessel development. vegfc expression was observed in the DA, as reported previously (Covassin et al., 2006), but no expression next to the intestinal vasculature was apparent (Fig. 8A,B). However, flt4 expression was observed in the SIV progenitors by in situ hybridization (Fig. 8C,D). We then analyzed SIV development by in situ hybridization of fli1a or confocal imaging of fli1a:EGFP in vegfcum18 (Villefranc et al., 2013), flt4hu4602 (Hogan et al., 2009b), and flt4sa9798 (Busch-Nentwich, 2013) mutant alleles. flt4hu4602 (expando) and vegfcum18 mutants have been previously reported to lack lymphangiogenesis. In addition, the expando mutant also lacks venous ISV sprouting. The flt4sa9798 allele contains a chemically induced nonsense point mutation (T>A) resulting in an early stop codon in an Ig domain (exon 12) necessary for dimerization of the receptor. We observed a complete loss of flt4 message by in situ hybridization in 8 out of 41 embryos (19.5%) from an incross of identified heterozygous fish suggesting that this allele is null for Flt4 function (Suppl. Fig. S5). In vegfc and both flt4 mutant alleles, we observed that the SIV formed normally in both homozygous mutant embyos as well as their heterozygous siblings (Fig. 8E–J)

Figure 8.

Figure 8

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vegfc and flt4 are dispensable for formation, but sufficient to induce ectopic growth of the SIV. (A,B) Based on in situ hybridization, vegfc expression is localized to the dorsal aorta, while flt4 shows expression in the progenitors of the intestinal vessels at 42 and 48 hpf (C,D). (E–H) In situ hybridization of vegfc−/−, flt4−/−, and sibling embryos for fli1a. (E,F) vegfc−/− embryos demonstrate normal SIV development at 52 hpf as compared to wild type and heterozygous siblings. Homozygous flt4sa9798 (G,H) and flt4hu4602 (I,J) embryos also have normal SIV development at 64 hpf and 52 hpf by in situ hybridization (G,H) and confocal microscopy of Tg(fli1a:GFP) embryos (I,J) as compared to wild-type and heterozygous siblings. (K,L) Heat shock induced overexpression of vegfc in hsp70:Vegfc embryos at 30 hpf results in hyperbranching and ectopic ventral sprouting of the SIV at 52 hpf as observed by confocal imaging of Tg(kdrl:mCherry). Large arrows indicate SIV. Anterior-left, dorsal-top.

To test the effect of vegfc overexpression on development of the SIV, we generated heat-shock inducible Tg(hsp70:Vegfc) line. Induction of vegfc by heat shock at 30 hpf, confirmed by in situ hybridization (Suppl. Fig. S6), resulted in hyperbranching and ectopic ventral sprouting of the SIV at 52 hpf (Fig. 8K,L) in addition to an increase in the number of parachordal lymphangioblast cells as previously reported (data no shown) (Cermenati et al., 2013; Le Guen et al., 2014). Intriguingly, heat-shock induced vegfc overexpression induced ectopic sprouting from SIV even in flt4 mutant embryos (Suppl. Fig. S7). These results suggest that although Vegfc/Flt4 signalling is not required for SIV development, vegfc overexpression can induce ectopic sprouting of the SIV.

Discussion

These results demonstrate that the endothelial progenitors of the sub-intestinal vein and the supra-intestinal artery migrate from the posterior cardinal vein and coalesce to form the new vessels. Vegfaa/Vegfr2 signaling is necessary for the formation of the intestinal vessels. vegfaa is expressed in the endoderm next to the site where SIA and SIV coalesce, and therefore it likely provides the guidance signal. Knockout of vegfaa resulted in failure of the sub-intestinal vein to develop properly. In addition, knockdown of the two zebrafish vegfr2 homologs, kdrl and kdr, resulted in a similar loss of SIV and SIA patterning. Knockout of Vegfc or Vegfr3 did not cause any significant defects in SIV formation. However, overexpression of vegfc did result in hyperbranching and ectopic growth. Together these results suggest that Vegfaa signaling pathway is responsible for the vascularization of the gut tube in zebrafish but other Vegf homologs such as Vegfc are sufficient to induce intestinal vascular development.

This study is one of the first detailed investigations of intestinal vascularization in zebrafish and gives insight into a previously uncharacterized mechanism of organ specific vascularization. Our model (Fig. 9) illustrates that differentiated endothelial cells migrate out of existing vasculature and coalesce to form new blood vessels. These new vessels do not retain connections to the original vessels as in branching or sprouting angiogenesis. Therefore the mechanism of intestinal vascularization is different from the standard definitions of vasculogenesis and angiogenesis. The coalescence of angioblasts into intestinal vasculature is similar to vasculogenesis, except that angioblasts are derived from the preexisting vessel, which is the defining feature of angiogenesis. It is also interesting to note that endothelial cells derived from a vein are able to form the arterial vessel SIA. Our results argue that SIA progenitors initially do not express arterial markers, consistent with their venous origin, and only upregulate arterial Notch reporter expression during later stages. Future studies are needed to determine if the mechanism of arterial-venous specification in the SIA and the SIV is similar to that in the DA and the PCV.

Figure 9.

Figure 9

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Model of zebrafish intestinal vascular development. Diagrams show progression of intestinal vasculature development. Beginning at approximately 24 hpf, cells begin to exit the PCV (A) and migrate ventrally in an anterior to posterior wave over the next 24 hours. These cells extend filopodia and coalesce to form the SIV by 56 hpf (B–D). Angiogenic sprouts which will give rise to the ICVs start extending from the SIV by 42–48 hpf (C) and continue extending through 56 hpf (D). Cells migrate in a second wave beginning at approximately 40 hpf to form the SIA with appearance of the stereotypical and functional vessels by 72 hpf (D,E).

It has been previously proposed that vasculature in many organs forms at least in part by vasculogenesis de novo. This was largely based on the observation in histological sections of individual angioblasts, which were not connected to the existing vessels, such as during liver and lung vascular development in mouse and chick embryos (Anderson-Berry et al., 2005; Kingsbury et al., 1956; Matsumoto et al., 2001; Sherer, 1991; Zeng et al., 1998). Based on our results, it is tempting to speculate that in other vertebrates, similar to zebrafish, organ specific vascularization also happens by the migration and coalescence of individual endothelial cells, which are derived from the existing vasculature. Therefore vasculogenesis de novo may not be the primary mechanism for organ vascularization.

Chemical treatment with Vegfr inhibitor SU5416 starting at 32 hpf completely prevented SIA and SIV formation. However, multiple other vascular defects such as inhibition of ISV angiogenesis were observed. This is consistent with a known Vegf requirement in angiogenesis and maintenance of blood vasculature. In contrast, treatment starting at 44 hpf had a more specific effect on the development of intestinal vasculature without greatly affecting the rest of the vasculature. This argues that intestinal vasculature is the most sensitive to Vegf requirement during these treatment stages and suggests that Vegf signaling is directly required for intestinal vessel development. This is supported by the Vegf receptor and vegfaa ligand mutant studies.

Multiple Vegf receptors, including kdrl, kdr and flt4 are expressed in the developing SIV. Our data indicate that vegfaa is expressed at the intestinal vasculature formation site, and mutation in vegfaa or loss of the primary Vegfaa receptors kdrl and kdr results in severe SIV and SIA formation defects. These results argue for the primary role of Vegfa / Vegfr2 signaling in intestinal vessel development. Interestingly, a partial development of SIV is still observed in a fraction of vegfaa mutant embryos. A knockdown of the other duplicate zebrafish Vegfa homolog, Vegfab, has been reported to result in reduced intestinal vessel development (Bahary et al., 2007). Therefore it is possible that both Vegfaa and Vegfab may function partially redundantly in the SIV and SIA formation. In contrast, loss of Vegfc or its receptor Flt4 does not result in any obvious defects in SIV or SIA formation suggesting that this signaling is dispensable for intestinal vessel formation. Although, there exists the possibility that the mutants used for this study are not fully null as vegfcum18 is still capable of signaling but is not secreted efficiently (Villefranc et al., 2013). However, this possibility is unlikely because there is no detectable flt4 mRNA present in flt4sa9798 mutants, and previous study indicated that flt4hu4602 represents a null or strong loss-of-function allele (Herbert et al., 2009; Hogan et al., 2009b). Interestingly, vegfc overexpression resulted in hyperbranching and ectopic sprouting from the SIV. This suggests that ectopic Vegfc signaling is sufficient to induce ectopic sprouting from SIV, while normally this function is performed by Vegfa/Vegfr2 homologs. Intriguingly, Vegfc overexpression induced ectopic sprouting even in flt4 mutant embryos. It is possible that flt4 mutant is not a complete null, and high level of Vegfc overexpression is sufficient to stimulate the remaining Flt4 activity. Alternatively, in some cases VEGFC has been reported to stimulate VEGFR2 expressing endothelial cells (Eichmann et al., 1998). It also has been suggested that zebrafish Kdrl belongs not to Vegfr2 but Vegfr4 subfamily, which has been lost in higher vertebrates (Bussmann et al., 2008), and therefore, differently from mammalian VEGFR2, it may be able to interact with Vegfc. We also cannot exclude the possibility that multiple Vegf ligands and receptors, including Kdr, Kdrl and Flt4 function redundantly during intestinal vascular development.

While this manuscript was in preparation, several other groups also reported the description of intestinal vessel development (Goi and Childs, 2015; Hen et al., 2015; Lenard et al., 2015; Nicenboim et al., 2015). In agreement with our results, time-lapse imaging and fate mapping using Kaede photoconversion confirmed that both SIV and SIA are derived from the PCV. Differently from Hen et al which reported that the SIA is derived from the SIV progenitor vessel (Hen et al., 2015), we find that the angioblasts from the PCV directly populate the SIA and SIV, and the SIA is already present when the interconnecting vessels (ICV) form. The SIA is located deep and very close to the PCV, therefore it is difficult to observe the early SIA progenitors using same promoter lines (fli1:dsRed; fli1:nGFP) which show uniform expression in the PCV and intestinal vasculature. In contrast, kdrl:mCherry line used in our study has very low expression in the intestinal vasculature, which allowed to differentiate between the PCV (high fli1:GFP and high kdrl:mCherry) and the early SIA progenitors (high fli1:GFP and low kdrl:mCherry). However, our results do not exclude the migration of some cells from the SIV to the SIA as reported by Hen et al, therefore it is likely that SIA forms initially via the direct migration of angioblasts from the PCV while some cells are added later when the ICVs form by migration from the SIV.

In summary, this study describes a novel mechanism during intestinal vascular development in zebrafish and demonstrates Vegfaa requirement in this process. This may serve as a model to understand how organ specific vasculature forms in other tissues and in different vertebrates.

Supplementary Material

1
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2
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3
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6

  • Zebrafish intestinal vasculature forms by a novel mechanism of angioblast migration.

  • Angioblasts migrate from the cardinal vein to form intestinal vessels.

  • Vegfaa provides a likely guidance signal for intestinal progenitor migration.

  • Kdr and Kdrl have partially overlapping roles in intestinal vessel development.

Acknowledgments

This research was supported by NIH R01 HL107369 award to S.S., the Max Planck Society (A.R. and D.Y.R.S.), and NIH T32 HL125204 award to A.L.K. We thank N. Lawson for providing kdrl and vegfc mutants and S. Lin for providing etv2:mCherry reporter line.

Footnotes

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Contributor Information

Andrew L. Koenig, Email: andrew.koenig@cchmc.org.

Kristina Baltrunaite, Email: kristina.baltrunaite@cchmc.org.

Neil I. Bower, Email: n.bower@imb.uq.edu.au.

Andrea Rossi, Email: andrea.rossi@mpi-bn.mpg.de.

Didier Y. R. Stainier, Email: didier.stainier@mpi-bn.mpg.de.

Benjamin M. Hogan, Email: b.hogan@imb.uq.edu.au.

Saulius Sumanas, Email: saulius.sumanas@cchmc.org.

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Supplementary Materials

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5
Download video file (4.5MB, mp4)
6
NIHMS752496-supplement-6.pdf (6MB, pdf)

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