Local angiogenic interplay of Vegfc/d and Vegfa controls brain region-specific emergence of fenestrated capillaries
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Curated by eLife
eLife assessment
This is a comprehensive approach to understanding the genetic requirements of the VEGF family in the generation of brain capillary structure in zebrafish. The manuscript combines vascular brain/meningeal anatomy revealed with a variety of reporter lines, with mutants for Wnt/β-catenin signaling and angiogenic cues. In particular revealing genetic redundancy in VEGF receptor family in an essential function in vasculature development.
This paper is significant because it provides compelling evidence that features methods, data, and analyses more rigorous than the current state-of-the-art in analysis of brain vasculature development. The data derived from the reporter lines are convincing, and the germline mutations allow for solid visualization and quantification of results.
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Abstract
Fenestrated and blood-brain barrier (BBB)-forming endothelial cells constitute major brain capillaries, and this vascular heterogeneity is crucial for region-specific neural function and brain homeostasis. How these capillary types emerge in a brain region-specific manner and subsequently establish intra-brain vascular heterogeneity remains unclear. Here, we performed a comparative analysis of vascularization across the zebrafish choroid plexuses (CPs), circumventricular organs (CVOs), and retinal choroid, and show common angiogenic mechanisms critical for fenestrated brain capillary formation. We found that zebrafish deficient for Gpr124, Reck, or Wnt7aa exhibit severely impaired BBB angiogenesis without any apparent defect in fenestrated capillary formation in the CPs, CVOs, and retinal choroid. Conversely, genetic loss of various Vegf combinations caused significant disruptions in Wnt7/Gpr124/Reck signaling-independent vascularization of these organs. The phenotypic variation and specificity revealed heterogeneous endothelial requirements for Vegfs-dependent angiogenesis during CP and CVO vascularization, identifying unexpected interplay of Vegfc/d and Vegfa in this process. Mechanistically, expression analysis and paracrine activity-deficient vegfc mutant characterization suggest that endothelial cells and non-neuronal specialized cell types present in the CPs and CVOs are major sources of Vegfs responsible for regionally restricted angiogenic interplay. Thus, brain region-specific presentations and interplay of Vegfc/d and Vegfa control emergence of fenestrated capillaries, providing insight into the mechanisms driving intra-brain vascular heterogeneity and fenestrated vessel formation in other organs.
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Author Response
Reviewer #1 (Public Review):
The manuscript of Parab et al. reports a beautiful phenotype analysis of the vascular brain/meningeal anatomy in a variety of reporter lines and mutants for Wnt/β-catenin signaling and angiogenic cues (Vegfaa, Vegfab Vegfc, Vegfd) during zebrafish development.
The present study extends the previous work of the same Parab, Quick, and Matsuoka, that focused on fenestrated vessel formation in the zebrafish myelencephalic choroid plexus (mCP). Vegfs were shown to regulate fenestrated vessel formation in combination, but not individually, and with only little effect on neighboring non-fenestrated brain vessel development. The fenestrated endothelium is thus known to have specific angiogenic requirements.The scale of investigation has now changed, and fenestrated vessel formation has been …
Author Response
Reviewer #1 (Public Review):
The manuscript of Parab et al. reports a beautiful phenotype analysis of the vascular brain/meningeal anatomy in a variety of reporter lines and mutants for Wnt/β-catenin signaling and angiogenic cues (Vegfaa, Vegfab Vegfc, Vegfd) during zebrafish development.
The present study extends the previous work of the same Parab, Quick, and Matsuoka, that focused on fenestrated vessel formation in the zebrafish myelencephalic choroid plexus (mCP). Vegfs were shown to regulate fenestrated vessel formation in combination, but not individually, and with only little effect on neighboring non-fenestrated brain vessel development. The fenestrated endothelium is thus known to have specific angiogenic requirements.The scale of investigation has now changed, and fenestrated vessel formation has been examined throughout the brain, in both circumventricular organs (organum vasculosum of lamina terminalis) and other choroid plexuses (CPs) including the diencephalic CP and its interface with the pineal gland, the eye choroid (choriocapillaris), and the hypophysis vasculature. The original finding is that a regionspecific code of angiogenic cues controls fenestrated vessel formation. The authors show that fenestrated vessels form independently of Wnt/β-catenin signaling and BBB vascular development but require different combinations of Vegfa and Vegfc/d-dependent angiogenesis within and across brain regions. A previously unappreciated function of autocrine and paracrine Vegfc signaling is demonstrated in this brain region-specific regulation of fenestrated capillary development.
Twenty-one different fish lines accurately genotyped and characterized and including a new Reck mutant, have been instrumental to conduct vascular pattern analysis, using confocal and stereomicroscopy imaging combined with transmission EM. High-quality illustration and robust quantification methods, previously validated, have been used. The study is well organized and reflects the high expertise and strong methodology of the investigators. Data are presented in nine dense figures and the contribution of angiogenic ligands to fenestrated vessel formation can hardly be studied more indepth.
However, and this will be my only main concern, no information is provided on the regional diversity of angiogenic receptor expression that may correlate with the regional angiogenic factor code. Without asking for a spatial transcriptomic study, the combination of Vegfr-reporter lines or in situ hybridization with a combination of receptor probes would allow for generating a comprehensive set of ligand/receptor data relative to the regional angiogenic signaling pattern involved in fenestrated vessel formation.
We appreciate this reviewer’s positive and encouraging comments highlighting both the quality and significance of our study. As we commented in response to the Essential Revisions point #1, we anticipate that a detailed expression analysis of all four Vegf receptors at different developmental stages during CP and CVO vascularization will be best addressed with new technologies combined with optimizations of existing tools/protocols. Thus, we have provided a paragraph of discussion on our perspectives for potential Vegf receptors involved in CP and CVO vascularization in the current study.
We address each of the points raised by the reviewer below.
Reviewer #2 (Public Review):
Building on their previous studies, Parab et al used a larger collection of genetically modified zebrafish lines to map the precise expression domains of different VEGF isoforms in the brain and demonstrated that different combinations of VEGF isoforms differentially control the formation of fenestrated vessels at different locations in the brain.
The authors used three Wnt signaling mutants to convincingly show wnt signaling is essential for parenchymal angiogenesis, but not required for fenestrated vessel development, such as those in choroid plexus, suggesting fenestrated vessel and barrier vessel are differentially regulated. The previous work from this group has established that VEGF isoforms are critical for myelencephalic choroid plexus development. In this study, they carefully documented the developmental vessel patterning in the diencephalic choroid plexus/pineal gland interface. They also documented the local expression pattern of VEGF isoforms with a set of BAC transgenic fish, together with the phenotype of a series of VEGF mutant fish, the data well support that different combinations of VEGF isoforms regulate fenestrated vessel development at different brain locations.
Given a larger temporal and spatial domain, VEGFs are critical for all forms of vessel development, there are potential redundancy mechanisms to maintain hemostasis of VEGF signaling, in this study, no data is provided to address whether LOF of one form of VEGF affects the expression of other isoforms.
This work provided detailed evidence of different isoform combinations of VEGF regulate formation/patterning of the fenestrated vessel at CP, OVLT, and NH in zebrafish. It will be interesting to follow in the mammalian system, how well these findings are conserved, for example, which isoform of VEGF is critical for vascular patterning during the developmental stages of the pineal gland? How VEGF isoforms participate in choroid plexus development at different ventricle regions and subsequence secretory function maintenance. However, these tasks are challenging without a good genetic tool to locally manipulate VEGF isoform expression during mammalian brain vessel development.
We appreciate this reviewer’s favorable and encouraging comments highlighting both the quality and impact of our study. We also acknowledge the great importance of the points raised by the reviewer, including the Vegf redundancy mechanisms and also our results’ conservation in mammals.
Reviewer #3 (Public Review):
Parab et al. investigate the requirement of specific Vegf ligands during the embryonic development of new blood vessels in different brain regions. The authors implement their previously published experimental paradigm (Parab et al 2021 eLife) combined with new transgenic and mutant zebrafish lines to show that vegf ligands (vegfaa, vegfab, vegfc, and vegfd) are required in various combinations to drive angiogenesis in distinct brain regions. Specifically, they show that individual loss of different vegf ligands causes either undetectable or partial effects in angiogenesis, while combined loss of vegf ligands results in severe defects in brain region-specific angiogenesis. As different blood vessel types (i.e. arteries, veins, lymphatics) require specific angiogenic cues, this study provides interesting new data on how the combination of these signals drives brain region-specific vascular development.
While the conclusions of the paper are generally well supported by the data, the authors overstate some of their findings, particularly with respect to the development of fenestrated capillaries. In this study, the authors use the zebrafish transgenic reporter line, plvap:EGFP, as an indicator of fenestrations. However, the authors do not provide any evidence of fenestrations of the blood vessels of the choroid plexuses or the cranial vessels used for quantification (Figures 1, 3, and 4). While expression of Plvap protein is often used as a marker for non-blood brain barrier endothelial cells, as Plvap is the major component of the diaphragms of fenestrated capillaries, plvap:EGFP expression alone does not indicate fenestrations. This is an important point because previous work has demonstrated that targeted deletion of Plvap does not cause a loss of fenestrations, but instead a loss of the diaphragms associated with fenestrations (Stan et al 2012 Dev Cell; Gordon et al 2019 Development). Similarly, Plvap expression alone does not necessarily indicate fenestrations as an expression of Plvap is not sufficient for fenestration formation. In fact, Plvap has initially been expressed in brain endothelial cells during initial angiogenesis to the brain without evidence of fenestrations, and subsequently, Plvap expression disappears during the maturation of the BBB. Thus, to conclude that specific vegf ligands are required for the development of fenestrated capillaries, transmission electron microscopy (TEM) should be used on the capillaries examined in this study or the language describing the results should be modified accordingly. Conversely, the authors did show TEM for the choriocapillaris (Figure 5A-C) but did not show plvap:EGFP expression in these vessels.
Additionally, the authors' usage of the phrase "development of fenestrated vessels" suggests that the study was examining signals that regulate the formation of fenestrations and not angiogenesis of vessels that may become fenestrated as demonstrated here. Therefore, as Plvap expression does not necessarily equate fenestrations (and vice-versa), the title and some of the major claims of the study are somewhat overstated.
We appreciate this reviewer’s constructive comments and suggestions to improve this study. We agree with the reviewer that the descriptions of our findings in the original manuscript were not strictly accurate in some aspects. We have now addressed the concern of the Tg(plvap:EGFP) reporter specificity by conducting additional molecular and functional characterizations of Tg(plvap:EGFP)+ vs Tg(glut1b:mCherry)+ brain vasculature, as we have commented in response to the Essential Revisions point #2. In addition, we have made substantial revisions in describing our findings, including 1) the change of the phrase "development of fenestrated vessels" into a more appropriate phrase and 2) the clarification of the primary focus of this manuscript on “angiogenesis/vascularization”. We believe that our revised manuscript now more clearly conveys the finding of signals involved in angiogenesis/vascularization of CP and CVO vascular beds.
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eLife assessment
This is a comprehensive approach to understanding the genetic requirements of the VEGF family in the generation of brain capillary structure in zebrafish. The manuscript combines vascular brain/meningeal anatomy revealed with a variety of reporter lines, with mutants for Wnt/β-catenin signaling and angiogenic cues. In particular revealing genetic redundancy in VEGF receptor family in an essential function in vasculature development.
This paper is significant because it provides compelling evidence that features methods, data, and analyses more rigorous than the current state-of-the-art in analysis of brain vasculature development. The data derived from the reporter lines are convincing, and the germline mutations allow for solid visualization and quantification of results.
-
Reviewer #1 (Public Review):
The manuscript of Parab et al. reports a beautiful phenotype analysis of the vascular brain/meningeal anatomy in a variety of reporter lines and mutants for Wnt/β-catenin signaling and angiogenic cues (Vegfaa, Vegfab Vegfc, Vegfd) during zebrafish development.
The original finding is that a region-specific code of angiogenic cues controls fenestrated vessel formation. The authors show that fenestrated vessels form independently of Wnt/β-catenin signaling and BBB vascular development but require different combinations of Vegfa and Vegfc/d-dependent angiogenesis within and across brain regions. A previously unappreciated function of autocrine and paracrine Vegfc signaling is demonstrated in this brain region-specific regulation of fenestrated capillary development.
My only main concern is that no information …
Reviewer #1 (Public Review):
The manuscript of Parab et al. reports a beautiful phenotype analysis of the vascular brain/meningeal anatomy in a variety of reporter lines and mutants for Wnt/β-catenin signaling and angiogenic cues (Vegfaa, Vegfab Vegfc, Vegfd) during zebrafish development.
The original finding is that a region-specific code of angiogenic cues controls fenestrated vessel formation. The authors show that fenestrated vessels form independently of Wnt/β-catenin signaling and BBB vascular development but require different combinations of Vegfa and Vegfc/d-dependent angiogenesis within and across brain regions. A previously unappreciated function of autocrine and paracrine Vegfc signaling is demonstrated in this brain region-specific regulation of fenestrated capillary development.
My only main concern is that no information is provided on the regional diversity of angiogenic receptor expression that may correlate with the regional angiogenic factor code. Without asking for a spatial transcriptomic study, the combination of Vegfr-reporter lines or in situ hybridization with a combination of receptor probes would allow for generating a comprehensive set of ligand/receptor data relative to the regional angiogenic signaling pattern involved in fenestrated vessel formation.
-
Reviewer #2 (Public Review):
Building on their previous studies, Parab et al used a larger collection of genetically modified zebrafish lines to map the precise expression domains of different VEGF isoforms in the brain and demonstrated that different combinations of VEGF isoforms differentially control the formation of fenestrated vessels at different locations in the 0brain.
The authors used three Wnt signaling mutants to convincingly show wnt signaling is essential for parenchymal angiogenesis, but not required for fenestrated vessel development, such as those in choroid plexus, suggesting fenestrated vessel and barrier vessel are differentially regulated. The previous work from this group has established that VEGF isoforms are critical for myelencephalic choroid plexus development. In this study, they carefully documented the …
Reviewer #2 (Public Review):
Building on their previous studies, Parab et al used a larger collection of genetically modified zebrafish lines to map the precise expression domains of different VEGF isoforms in the brain and demonstrated that different combinations of VEGF isoforms differentially control the formation of fenestrated vessels at different locations in the 0brain.
The authors used three Wnt signaling mutants to convincingly show wnt signaling is essential for parenchymal angiogenesis, but not required for fenestrated vessel development, such as those in choroid plexus, suggesting fenestrated vessel and barrier vessel are differentially regulated. The previous work from this group has established that VEGF isoforms are critical for myelencephalic choroid plexus development. In this study, they carefully documented the developmental vessel patterning in the diencephalic choroid plexus/pineal gland interface. They also documented the local expression pattern of VEGF isoforms with a set of BAC transgenic fish, together with the phenotype of a series of VEGF mutant fish, the data well support that different combinations of VEGF isoforms regulate fenestrated vessel development at different brain locations.
Given a larger temporal and spatial domain, VEGFs are critical for all forms of vessel development, there are potential redundancy mechanisms to maintain hemostasis of VEGF signaling, in this study, no data is provided to address whether LOF of one form of VEGF affects the expression of other isoforms.
This work provided detailed evidence of different isoform combinations of VEGF regulate formation/patterning of the fenestrated vessel at CP, OVLT, and NH in zebrafish. It will be interesting to follow in the mammalian system, how well these findings are conserved, for example, which isoform of VEGF is critical for vascular patterning during the developmental stages of the pineal gland? How VEGF isoforms participate in choroid plexus development at different ventricle regions and subsequence secretory function maintenance. However, these tasks are challenging without a good genetic tool to locally manipulate VEGF isoform expression during mammalian brain vessel development.
-
Reviewer #3 (Public Review):
Parab et al. investigate the requirement of specific Vegf ligands during the embryonic development of new blood vessels in different brain regions. The authors implement their previously published experimental paradigm (Parab et al 2021 eLife) combined with new transgenic and mutant zebrafish lines to show that vegf ligands (vegfaa, vegfab, vegfc, and vegfd) are required in various combinations to drive angiogenesis in distinct brain regions. Specifically, they show that individual loss of different vegf ligands causes either undetectable or partial effects in angiogenesis, while combined loss of vegf ligands results in severe defects in brain region-specific angiogenesis. As different blood vessel types (i.e. arteries, veins, lymphatics) require specific angiogenic cues, this study provides interesting new …
Reviewer #3 (Public Review):
Parab et al. investigate the requirement of specific Vegf ligands during the embryonic development of new blood vessels in different brain regions. The authors implement their previously published experimental paradigm (Parab et al 2021 eLife) combined with new transgenic and mutant zebrafish lines to show that vegf ligands (vegfaa, vegfab, vegfc, and vegfd) are required in various combinations to drive angiogenesis in distinct brain regions. Specifically, they show that individual loss of different vegf ligands causes either undetectable or partial effects in angiogenesis, while combined loss of vegf ligands results in severe defects in brain region-specific angiogenesis. As different blood vessel types (i.e. arteries, veins, lymphatics) require specific angiogenic cues, this study provides interesting new data on how the combination of these signals drives brain region-specific vascular development.
While the conclusions of the paper are generally well supported by the data, the authors overstate some of their findings, particularly with respect to the development of fenestrated capillaries. In this study, the authors use the zebrafish transgenic reporter line, plvap:EGFP, as an indicator of fenestrations. However, the authors do not provide any evidence of fenestrations of the blood vessels of the choroid plexuses or the cranial vessels used for quantification (Figures 1, 3, and 4). While expression of Plvap protein is often used as a marker for non-blood brain barrier endothelial cells, as Plvap is the major component of the diaphragms of fenestrated capillaries, plvap:EGFP expression alone does not indicate fenestrations. This is an important point because previous work has demonstrated that targeted deletion of Plvap does not cause a loss of fenestrations, but instead a loss of the diaphragms associated with fenestrations (Stan et al 2012 Dev Cell; Gordon et al 2019 Development). Similarly, Plvap expression alone does not necessarily indicate fenestrations as an expression of Plvap is not sufficient for fenestration formation. In fact, Plvap has initially been expressed in brain endothelial cells during initial angiogenesis to the brain without evidence of fenestrations, and subsequently, Plvap expression disappears during the maturation of the BBB. Thus, to conclude that specific vegf ligands are required for the development of fenestrated capillaries, transmission electron microscopy (TEM) should be used on the capillaries examined in this study or the language describing the results should be modified accordingly. Conversely, the authors did show TEM for the choriocapillaris (Figure 5A-C) but did not show plvap:EGFP expression in these vessels.
Additionally, the authors' usage of the phrase "development of fenestrated vessels" suggests that the study was examining signals that regulate the formation of fenestrations and not angiogenesis of vessels that may become fenestrated as demonstrated here. Therefore, as Plvap expression does not necessarily equate fenestrations (and vice-versa), the title and some of the major claims of the study are somewhat overstated.
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