Neurovascular Communication during CNS Development

Developmental Cell

Review Neurovascular Communication during CNS Development Isidora Paredes,1,2,3 Patricia Himmels,1,2,3 and Carmen Ruiz de Almodo´var1,2,* 1Biochemistry

Center, Heidelberg University, 69120 Heidelberg, Germany Center for Neurosciences, Heidelberg University, 69120 Heidelberg, Germany 3These authors contributed equally *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2018.01.023 2Interdisciplinary

A precise communication between the nervous and the vascular systems is crucial for proper formation and function of the central nervous system (CNS). Interestingly, this communication does not only occur by neural cells regulating the growth and properties of the vasculature, but new studies show that blood vessels actively control different neurodevelopmental processes. Here, we review the current knowledge on how neurons in particular influence growing blood vessels during CNS development and on how vessels participate in shaping the neural compartment. We also review the identified molecular mechanisms of this bidirectional communication. Introduction The vertebrate CNS is a complex, highly organized structure, responsible for sending, receiving, integrating, and processing information from all parts of the body. It is composed of multiple cell types (e.g., neurons, astrocytes, microglia, blood vessels (endothelial cells [ECs] lining the vessel wall and mural cells covering ECs), oligodendrocytes, etc.) that communicate to each other to ensure proper development and functionality of the system. Research over the past decade showed that the vascular and the nervous systems share common guidance cues for their development and patterning (Adams and Eichmann, 2010; Eichmann and Thomas, 2013; Quaegebeur et al., 2011; Tam and Watts, 2010; Walchli et al., 2015). It also showed that the cellular morphological structures of the axon growth cone (structure located at the leading edge of a growing axon) and the endothelial tip cell (EC located at the tip of a sprouting blood vessel) are alike and respond to growth and guidance signals in similar ways (Walchli et al., 2015). Classical axon guidance molecules (Netrins, Slits, Semaphorins, and Ephrins), as well as members of the vascular endothelial growth factor (VEGF) family (VEGF-A, -B, -C, and -D) were identified as common signals regulating both vessel and neuronal development. As excellent articles have already reviewed these studies (Adams and Eichmann, 2010; Eichmann and Thomas, 2013; Quaegebeur et al., 2011; Walchli et al., 2015), they will not be discussed here and we refer to them for further insights. Depending on the organ where they reside, ECs of the CNS possess different molecular and functional properties, giving rise to the vascular heterogeneity observed within an organism (Augustin and Koh, 2017; Potente and Makinen, 2017). To serve the needs of the nervous tissue, CNS blood vessels are highly specialized. CNS ECs have unique characteristics, as they lack fenestration, are interconnected by tight and adherens junctions, and express specific molecular transporters (Daneman and Prat, 2015). These characteristics build up the blood brain barrier (BBB), which seals the CNS and controls substance influx and efflux. Pericytes and astrocytic endfeet surround ECs and provide functional support for the BBB via intercellular communication mechanisms. EC-specific characteristics are induced and maintained by the neural tissue and already appear during 10 Developmental Cell 45, April 9, 2018 ª 2018 Elsevier Inc.

CNS vascularization, which starts at embryonic stages (Daneman and Prat, 2015; Hupe et al., 2017). Blood vessel growth and maturation in the CNS occurs at the same time as different neural cell types are generated and circuits are established. This concomitant development, and the specific properties of CNS blood vessels, raises important questions: Are their development and branching patterns established independently or coordinately? How do neurons instruct blood vessel formation according to the requirements of CNS development? Do vessels also actively signal to neural cells? Here we particularly review the mechanisms of how neurons communicate with blood vessels during embryonic and postnatal development, by focusing on the brain, spinal cord, and retina. We also emphasize how blood vessels are not just conductors that transport oxygen and nutrients but how they also have scaffold and paracrine signaling functions to modulate the development and function of different neural cell populations. Neural and Vascular Structures Develop, Grow, and Mature Simultaneously The development of the vertebrate CNS begins early during embryogenesis. At around mouse embryonic day 7.5 (E7.5), the neural plate establishes and starts folding to form the neural tube. The neural tube becomes then regionalized along the anteroposterior axis (it divides into the rostral primary brain vesicles [forebrain, midbrain, and hindbrain] and the caudal spinal cord). Subsequently, by E9.5 dorsoventral patterning of neural tube progenitors is established (Dessaud et al., 2008) and progenitor proliferation, differentiation, and migration of differentiated neurons and glia start taking place. Notably, these latter processes happen simultaneously with the development of the CNS vasculature. The first step of CNS vascularization is the formation of the perineural vascular plexus (PNVP) around the neural tube (Figures 1A and 1B), which in mice occurs between E8.5 and E10 by the assembly of somite-derived angioblasts into a blood vessel network (Hogan et al., 2004). Subsequently, the developing brain and spinal cord will become vascularized by secondary angiogenesis from the PNVP, where new vessel sprouts will invade the CNS and extend toward the ventricle (Kurz, 2009;

Developmental Cell

Review A

B

C

Figure 1. CNS Vascularization Processes (A) Neocortical development. Neurogenesis begins around E9 to E10 in the germinal zone of the mouse cortex. During neurogenesis, NSCs/NPCs give rise to neuroblasts, and eventually to neurons, OLPs, OLs, and astrocytes. Simultaneously, the PNVP forms. Subsequently, sprouting vessels from the PNVP start (legend continued on next page)

Developmental Cell 45, April 9, 2018 11

Developmental Cell

Review Tata et al., 2015). Vessels reaching the ventricle give rise to new branches that surround the ventricle and reverse their direction toward the pia. Finally, branches anastomose with other branches, forming a rich capillary plexus (Figure 1A). Interestingly, vascularization of the embryonic forebrain (telencephalon) does not only occur by sprouting blood vessels from the PNVP but also from a periventricular vascular plexus, which originates from a prominent basal vessel located in the basal ganglia primordium, independently of the PNVP (Vasudevan et al., 2008). This vascularization process seems to be controlled by ventral and dorsal homeobox transcription factors expressed in ECs (Vasudevan et al., 2008). As part of the CNS, the mouse retina is also commonly used to study mechanisms of angiogenesis. The retina starts its development at E8.5 to E9.0 and finalizes postnatally. Newborn postmitotic neurons are generated in an ordered but overlapping temporal manner conferring the neural retina a laminar organization (Heavner and Pevny, 2012). Within the first postnatal weeks, lamination of the retina (completed around P7) as well as dendritogenesis and synaptogenesis of retinal neurons occur (Fan et al., 2016). During the same postnatal time frame, the retina vasculature develops. From postnatal day 0 (P0) to P8 a superficial vascular primary plexus, guided by a previously formed mesh-like astrocyte network, expands by radial outgrowth of vessel sprouts from the optic nerve to the periphery (Selvam et al., 2017). From P7, vessels from the superficial layer sprout vertically into the plexiform layers to form parallel and interconnected networks, termed deep and intermediate vascular plexus (Figure 1C). The three retinal networks mature and actively irrigate the retina during the third postnatal week (Selvam et al., 2017). The formation of the BBB occurs concomitantly with blood vessel growth into the CNS. For further insights on BBB formation, we refer to recent reviews on this topic (Daneman and Prat, 2015; Zhao et al., 2015). Specific Molecular Mechanisms Controlling CNS Vascularization Research over the past years showed that neural progenitors, differentiated neurons, and glia play a fundamental role in CNS vascularization. As one of the key angiogenic factors, VEGF is crucial in this process (Hogan et al., 2004; Mackenzie and Ruhrberg, 2012). Supporting the concept of CNS-specific blood vessels and vascularization mechanisms, recent research demonstrates that vascularization of the CNS is controlled, together with classical angiogenic factors such as VEGF, by CNS-specific

vascular cues (Ruhrberg and Bautch, 2013; Tata et al., 2015) (Table 1). Neuroepithelium-derived Wnt7a/b (leading to canonical Wnt signaling in CNS ECs) and the G-protein coupled receptor GPR124, expressed in ECs, are specifically required for CNS vascularization (Anderson et al., 2011; Cullen et al., 2011; Kuhnert et al., 2010). Although initially identified as separated molecular mechanisms, the striking similarities between the phenotype of Wnt7a/b double mutants and GPR124 mutant embryos led to the hypothesis that both signaling mechanisms might converge into a common pathway in ECs. These phenotypes comprised normal PNVP formation but blunted angiogenesis with glomeruloid structures in the forebrain and ventral spinal cord, together with severe CNS-specific hemorrhages and embryonic lethality at E12.5 (Anderson et al., 2011; Cullen et al., 2011; Daneman et al., 2009; Kuhnert et al., 2010; Stenman et al., 2008). Indeed, three following studies linked the mechanisms by identifying that GPR124 serves as a Wnt7-specific co-activator of canonical Wnt signaling in ECs (Posokhova et al., 2015; Vanhollebeke et al., 2015; Zhou and Nathans, 2014). Recently, RECK (reversion-inducing cysteine-rich protein with Kazal motifs; a GPI-anchored membrane protein) has been described as an additional co-receptor for Wnt7a/b in CNS ECs, required for proper Wnt7a/b signaling during CNS angiogenesis and establishment of BBB properties (Cho et al., 2017; Ulrich et al., 2016; Vanhollebeke et al., 2015). Interestingly, and supporting the idea that the molecular mechanisms that control CNS vascularization can be regional-dependent, a distinct set of Wnt ligands, receptors, and co-receptors control €ller vascular development in the developing retina. There, the Mu glia-derived ligand Norrin, together with the receptor Frizzled4 (Fz4), co-receptor Lrp5, and co-activator Tspan12, mediates Wnt/b-catenin signaling to control angiogenesis, blood-retina barrier formation, and maintenance (Junge et al., 2009; Lai et al., 2017; Xu et al., 2004; Ye et al., 2009). Wnt/b-catenin signaling in brain ECs regulates the expression of the death receptors DR6 and TROY, which are enriched in brain vasculature and were described to act as regulators of CNS-specific angiogenesis (Tam et al., 2012). Altogether, these studies suggest that GPR124/Wnt signaling in CNS ECs results in a specific transcriptional program, providing a further layer of regulation for CNS vascularization. Whether these newly identified pathways converge in the VEGF signaling pathway to further confer specificity to VEGF-induced angiogenesis in the CNS is a question that remains to be answered. In this respect, although further in vivo proof is lacking, in vitro experiments show that

extending radial branches from the plexus toward the ventricle. These branches give rise to new branches, which anastomose and form a highly wired vascular network. NSC/NPC, neural stem cell/neural progenitor cell; OLP, oligodendrocyte precursor; OL, oligodendrocyte. (B) Spinal cord development. At around E7.5 to E8.5 in mice, mesodermal angioblasts are recruited by NPCs to the periphery of the neural tube/spinal cord and form the PNVP. At the same time, neural tube progenitors are arranged along the dorsoventral axis in specific domains. At E9.5, MNs start to differentiate, and locate at the ventro-lateral side of the spinal cord. Between E9.5 and E10.5, vessel sprouts start invading radially the primitive spinal cord. Blood vessel sprouts follow a stereotypical growth pattern: regions as MN soma area (MN columns) and FP are left avascular during a developmental time window (until E12.5). At E12.5, additional capillaries grow and branch into MN columns, as well as extend a network all over the spinal cord. FP, floor plate; NPC, neural progenitor cell; MN, motor neuron; PNVP, perineural vascular plexus; RP, roof plate. (C) Postnatal retina development. Schematic representation of retinal vascular development between E20 and P15. Top: Overview of a whole flat mount retina. Bottom: Cross-section view of the retina layers. At E20, an astrocytic network starts forming over the neuroretina, invading from the optic nerve radially to the periphery while the retina is completely avascular. After birth, the astrocyte-forming network serves as a template for the invasion of pioneer ECs from the optic nerve. From P1 to P8 a superficial primary vascular plexus layer is formed above the retina ganglion cell layer (RGCL). Around P7, sprouts from the primary plexus start invading the neuroretina layers forming the deep vascular plexus located between the inner (INL) and the outer nuclear layer (ONL). A tertiary intermediate vascular plexus is formed at the upper boundary of the INL between P14 and P20. RPE, retinal pigment epithelium.

12 Developmental Cell 45, April 9, 2018

Neural-Derived Signal

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines/Genetic Approaches used in the Study

Comments

Reference

Developing Brain and Spinal Cord

Developmental Cell 45, April 9, 2018 13

NPCs

VEGF

ECs (not specified)

mouse/neural tube/spinal cord (E8.5–E12.5) quail/ neural tube (HH16–HH27)

NSE-VEGFtg

PNVP formation and blood vessel sprouting into the CNS parenchyma is promoted by VEGF signaling

(Himmels et al., 2017; Hogan et al., 2004; James et al., 2009)

NPCs (radial glia)

Vegfab

ECs (not specified)

zebrafish/spinal cord (76 hours post fertilization [hpf] to 30 days post fertilization [dpf])

TgBAC(gfap:gal4ff) Tg(gfap:NTR) Tg(elavl3:NTR) Tg(mbpa:NTR) irf8 mutants Tg(hsp70l:sflt1) Tg(hsp70l:sflt4) vegfaabns1 mutants vegfabbns92 mutants vegfchu6410 mutants flt1bns29; vegfabbns92 double mutants

CNS-resident radial glia control PNVP formation via Vegfab/Vegfr2 signaling

(Matsuoka et al., 2017)

NPCs

Wnt7a/b

ECs (not specified)

mouse/forebrain and ventral spinal cord (E10.5–E12.5)

Tie2-cre x b-cateninflox/flox Wnt7a+/ ; Wnt7b+/ Wnt7a / ; Wnt7b+/ Wnt7a+/ ; Wnt7b / Wnt7a / ; Wnt7b /

alterations of Wnt/b-catenin signaling in ECs leads to reduced vessels and formation of hemorrhagic vascular malformations as well as to changes of BBBspecific properties

(Daneman et al., 2009)

NPCs

not specified

ECs (not specified)

mouse/forebrain and spinal cord (E10.5–P0)

Gpr124Lz/Lz

deletion of Gpr124 leads to CNS-specific vascular defects characterized by delayed vascular sprouting and formation of glomeruloid tufts due to altered TGF-b signaling in ECs

(Anderson et al., 2011)

NPCs

Norrin (Ndp)

ECs (not specified)

mouse/hindbrain and ventral spinal cord (E11.5–P6)

Gpr124 / ; Ndp / Pdgfb-creERflox/flox x Gpr124flox/ ; Ndp / Gpr124 / ; Fz4 /

Norrin/Fz4 signaling plays a complementary and/or partial redundant role in Wnt/ Gpr124 signaling in CNS vascular development and BBB integrity

(Zhou and Nathans, 2014)

NPCs

Wnt7a/b

ECs (not specified)

mouse/forebrain and ventral spinal cord (E13.5)

GPR124+/ ; Wnt7a Wnt7b+/

CNS-specific developmental angiogenesis in the forebrain and ventral spinal cord is regulated by GPR124 acting as a Wnt7a/b-specific coactivator in ECs

(Posokhova et al., 2015)

/

;

(Continued on next page)

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Neural Cell Type

Vascular Cell Type (Requirement of Direct Contact with Blood Vessel)

Review

Table 1. Neural-Derived Signals Controlling CNS Angiogenesis during Development

14 Developmental Cell 45, April 9, 2018

Table 1.

Neural Cell Type

Continued

Neural-Derived Signal

Vascular Cell Type (Requirement of Direct Contact with Blood Vessel)

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines/Genetic Approaches used in the Study y72

Comments

Reference

ECs

zebrafish/hindbrain (48–96 hpf)

reck mutants Tg(hsp70l:Xla. TCFDC-EGFP) Tg(hsp70l:cab-catenin-2ATFP)W130

intracerebral vascularization, as well as expression of BBB-related markers, requires EC Reck activity in a noncell autonomous manner

(Ulrich et al., 2016)

NPCs

Wnt7a/b

ECs (not specified)

mouse/brain (LGE, MGE, cortex, hindbrain) and spinal cord (E11.5–P0)

Tie2-Cre x ReckDflex2/Dex1 ReckDex2/Dex2 Tie2-Cre x ReckDflex2/Dex2 Tie2-Cre x ReckDflex2/Dex2; NdpD/Y Pdgfb-Cre x ReckDfex2/Dex1; NdpD/Y Tie2-Cre x Reckflex2/Dex2; Gpr124fl/+ Tie2-Cre x Reckflex2/+; Gpr124fl/D Tie2-Cre x ReckDflex2/Dex2; Gpr124fl/D ReckCr/Dex2; Gpr124+/D

Reck and Gpr124 are an integral part of the cell surface protein complex that transduces Wnt7a/Wnt7bspecific signaling in mammalian CNS ECs, thereby promoting angiogenesis and BBB formation

(Cho et al., 2017)

OLPs

Wnt7a/b

ECs (not specified)

mouse/brain (corpus callosum) (P4-P11)

Plp-creERT2 x VHLflox/flox Sox10-cre x VHLflox/flox Olig1-cre x HIF1aflox/flox Olig1-cre x HIF2aflox/flox

OLP-intrinsic HIF1/2a signaling couples postnatal myelination and vessel growth via the expression of Wnt7a/b that signals in OLPs and in ECs

(Yuen et al., 2014)

NPCs

not specified

ECs (not specified)

mouse/brain (telencephalon) (E13.5–E14.5)

Foxg1-cre x Tgfbr2flox/flox

altered expression and/or localization of pro- and antiangiogenic factors due to neural deletion of Tgfbr2 leads to reduced branching and hampered EC migration

(Hellbach et al., 2014)

NPCs

not specified

ECs (not specified)

mouse/brain (telencephalon) (E10.5–E14.5)

Itgb8DNE Tgfb1 / Pdgfr-iCreERT2 x Tgfbr2 Alk5iDEC

avb8 integrin in NPCs activates TGF-b1 in a ventral-dorsal gradient in the brain, leading to signaling in ECs via Tgfbr2-ALK5Smad3, suppressing sprouting angiogenesis and thereby stabilizing blood vessels

(Arnold et al., 2014)

(Continued on next page)

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not specified

Review

Not specified

Developmental Cell 45, April 9, 2018 15

Neural Cell Type

Neural-Derived Signal

Vascular Cell Type (Requirement of Direct Contact with Blood Vessel)

Comments

Reference

NPCs

not specified

ECs (not specified)

mouse/brain (E13.5–E16.5) zebrafish/brain (1–4 dpf)

Alk1-cre x Nrp1flox/flox Nestin-cre x b8flox/flox ItgB8 morpholino in zebrafish Nrp1a morpholino in zebrafish Nrp1a + ItgB8 morpholino in zebrafish

activated avb8 integrin in NPCs fine-tunes sprouting cerebral angiogenesis by activating latent TGF-b, which then signals in ECs

(Hirota et al., 2015)

NPCs (radial glia)

TGF-b1

ECs (not specified)

mouse/brain (cortex) (E14–E16)

intraventricular injection of TGF-b1; intraventricular injection and electroporation in utero of shRNA-TGF-b1; in vitro studies

TGF-b1 signaling pathway is a potential mediator of the interactions of radial glial cells and ECs, thereby controlling blood vessel branching in the cerebral cortex

(Siqueira et al., 2017)

Neurons

Nogo-A

ECs (not specified)

mouse/brain (cortex, hippocampus, superior colliculus, corpus callosum) (P4–P10)

Nogo-A

Nogo-A knockout mice display increased number of capillaries and capillary branch points

(Walchli et al., 2013; Walchli et al., 2017)

NPCs

not specified

ECs (yes)

mouse/brain (cerebral cortex) (E14.5–E17.5)

Nestin-creER x orc3flox/flox hGFAP-cre x orc3flox/flox

ablation of radial glia results in vessel regression due to alterations in Wnt signaling in ECs and MMP-2 activity

(Ma et al., 2013)

OLPs

not specified

ECs (yes)

mouse/brain (telencephalon) (E12.5–E18.5)

Nkx2.1-cre x Rosa26-DTA Cspg4-cre x Rosa26-DTA

ablation of OLPs (NG2+ glia) results in severe reduction of blood vessel ramifications and connections by E18.5

(Minocha et al., 2015)

Astrocytes

not specified

ECs (yes)

mouse/brain (cortex) (P3–P7)

hGFAP-cre x orc3flox/flox

reduction of astroglia delays vessel growth and branching

(Ma et al., 2012)

Astrocytes

VEGF

ECs (not specified)

mouse/brain (RMS) (P3-P21)

wild-type (WT) mice

blood vessel formation and pattern along the RMS is promoted by VEGF signaling

(Bozoyan et al., 2012)

Neurons

VEGF/sFlt1

ECs (not specified)

mouse/spinal cord (E9.5–E12.5) chick/spinal cord (HH19–HH31)

NSE-VEGFtg in ovo RNAi (mi-sFlt1 and mi- NRP1)

proper blood vessel patterning around MN columns of the developing spinal cord is controlled by VEGF, with VEGF levels being fine-tuned via a NRP1sFlt1-VEGF signaling axis

(Himmels et al., 2017)

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines/Genetic Approaches used in the Study

/

(Continued on next page)

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Continued

Review

Table 1.

16 Developmental Cell 45, April 9, 2018

Table 1.

Neural Cell Type

Continued

Neural-Derived Signal

Vascular Cell Type (Requirement of Direct Contact with Blood Vessel)

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines/Genetic Approaches used in the Study ka601

Comments

Reference

Neurons

VEGF/sFIt1

ECs (not specified)

zebrafish/spinal cord (26 hpf–4 dpf)

flt1 mutants flt1ka605 mutants vhlhu2114 mutants flt1ka601; vhihu2114 neuronal-specific sFlt1 miRNA neuronal-specific and inducible Vegfaa overexpression

neuron-specific loss of Flt1, neuronal-specific knockdown of sFlt1 or increased neuronal Vegfaa expression results in venous hypersprouting around the developing spinal cord. The combination of Flt1 mutants with mutants that overexpress Vegfaa (flt1ka601;vhlhu2114) results in premature blood vessel sprouting into the spinal cord

(Wild et al., 2017)

NPCs (radial glia)

VEGF/sFlt1

ECs (not specified)

zebrafish/spinal cord (30 hpf-154 hpf)

Tg(gfap:NTR) Tg(elavl3:NTR) flt1bns29 mutants flt1fh390 mutants

blood vessel patterning around the spinal cord is controlled by EC-derived sFlt1 titration of VEGF. NPCs control sFlt1 expression in ECs and NPC ablation results in venous hypersprouting around the developing spinal cord

(Matsuoka et al., 2016)

NPCs

VEGF

ECs (not specified)

chick/spinal cord (E4–E5)

Gain of function (sFlt1, VEGF)

overexpression of sFlt1 in the spinal cord results in avascular areas

(Takahashi et al., 2015)

Developing Retina ECs (not specified)

mouse/retina (E17.5–P28)

Pax6a-Cre x Vegfr2flox/flox VEcad-CreERT2 x Vegfr2flox/flox Vav1-iCre x Vegfr2flox/flox

VEGFR2 expressed on retinal neurons, titrates VEGF in the retina, and thereby determines the direction of vascular growth

(Okabe et al., 2014)

RGCs

angiogenic factors VEGF

ECs (not specified)

rat/mouse/retina (P4–P15)

Six3-cre x Brn3bZ-dta/+

hypoxic retinas accumulate succinate, which is sensed by RGC receptor GPR91, in turn, they secrete VEGF and angiogenic factors that promote angiogenesis

(Sapieha et al., 2008)

(Continued on next page)

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VEGF

Review

Retinal neurons

Neural-Derived Signal

Vascular Cell Type (Requirement of Direct Contact with Blood Vessel)

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines/Genetic Approaches used in the Study flox/flox

Comments

Reference

Developmental Cell 45, April 9, 2018 17

Neurons

Slit2

ECs (not specified)

mouse/retina (P7–P17)

CAG:Slit2 CAG:Slit2flox/floxSlit1 / Robo 1 / Robo2flox/flox Robo1 / Robo2DEC Robo2flox/flox Robo4 /

Slit2, expressed by inner nuclear layer neurons, promotes retinal angiogenesis via Robo1 and Robo2 expressed on retinal ECs

(Rama et al., 2015)

Amacrine Horizontal neurons

VEGF

ECs (not specified)

mouse/retina (P13–P23)

Ptfa1-cre x Vhlflox/floc Ptfa1-cre x Hif1aflox/floc Ptfa1-cre x Hif2aflox/floc Ptfa1-cre x VEGFflox/floc

VEGF derived from inner nuclear layer neurons is required to develop and maintain intraretinal vasculature

(Usui et al., 2015)

Astrocytes

fibronectin

ECs (not specified)

mouse/retina (P0-P7)

Tlx

proangiogenic astrocytes form a fibronectin scaffold directed by hypoxia-driven Tlx, required for angiogenesis

(Uemura et al., 2006)

Astrocytes

VEGF

ECs (yes)

mouse/retina (P0–P15)

VEGFa120/120 VEGFa188/188 GFAP-PDGF-Atg aA-Crystallin- VEGF120tg aA-Crystallin- VEGF164tg aA-Crystallin- VEGF188tg

astrocytes and their derived VEGF guide endothelial tip cells and angiogenic sprouts during developmental angiogenesis

(Gerhardt et al., 2003)

Astrocytes

not specified

ECs (not specified)

mouse/retina (P5–P7)

GFAP-cre x Hif1 aflox/floc GFAP’Cre x Hif2aflox/floc GFAP-cre x Vhlflox/floc GFAP-cre x VEGFflox/floc

astrocyte-derived VEGF and its upstream regulators HIF1a and 2a are not required for developmental angiogenesis, but necessary for pathological hypoxic conditions

(Weidemann et al., 2010)

Astrocytes

VEGF

ECs (not specified)

mouse/retina (P5–P12)

GFAP-cre x Hif1aflox/floc GFAP -cre x VEGFflox/floc

astrocyte-derived VEGF has a minor role during developmental angiogenesis

(Scott et al., 2010)

€ller glia Mu

VEGF

ECs (not specified)

mouse/retina (P7–P17)

€ller glia-cre x VEGFflox/floc Mu

€ller glia-derived VEGF has Mu no defects in developmental angiogenesis, but on pathological hypoxic conditions

(Bai et al., 2009)

/

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Neural Cell Type

Continued

Review

Table 1.

Developmental Cell

Review DR6 and TROY are required for VEGF-mediated JNK activation and sprouting in human brain ECs (Tam et al., 2012). Apart from GPR124, other mechanisms have been found to modulate Wnt signaling in ECs during brain development. Retinoic acid ensures the formation of an adequate and stable brain vascular plexus during embryogenesis by suppressing the expression of Wnt inhibitors in neocortical progenitors and by a cell-autonomous modulation of Wnt signaling and its downstream target Sox17 in ECs (Bonney et al., 2016). Once the primitive vascular network is formed in the late embryonic cortex, vessel stabilization takes place at the same time as other vessel branches continue growing. Here, neural progenitors of the cerebral cortex have been shown to downregulate canonical Wnt signaling in ECs and thereby stabilize newly formed blood vessels (Ma et al., 2013). A recent study relates myelination with Wnt signaling and angiogenesis in the corpus callosum at P10 (at a time of robust angiogenesis in the CNS). It describes that hypoxia-inducible factor-1/2a (HIF1/2a) activity in oligodendrocyte precursors (OLPs) inhibits myelination by inducing the expression and autocrine signaling of Wnt7a/b (Yuen et al., 2014). At the same time, this OLP-derived Wnt7a/b acts on ECs to promote vessel growth (Yuen et al., 2014) (see Figure 4). Altogether, these studies indicate that for proper CNS vascularization in the developing CNS, Wnt signaling is temporally and dynamically regulated in ECs by different CNS cell types and molecular mechanisms. Although transforming growth factor b (TGF-b) also regulates angiogenesis in other organs apart from the CNS (ten Dijke and Arthur, 2007), its signaling can be specifically activated in the CNS during development by neural progenitors. There, b8 integrin, expressed in neural progenitors, activates extracellular matrix-bound latent TGF-b, which then signals to ECs (Arnold et al., 2014; Hirota et al., 2015; Nguyen et al., 2011). The activation of latent TGF-b is further regulated by EC-derived Neuropilin-1 (NRP1), which forms intercellular protein complexes with neural progenitor-derived b8 integrin. Cell type-specific inactivation of b8 integrin, NRP1, or TGF-b receptors results in cerebral angiogenesis defects due to impaired TGF-b signaling (Hirota et al., 2015; Nguyen et al., 2011). Consistently, endothelial expression of TGF-b type II receptor (Tgfbr2) is required for EC migration during retina vascularization (Allinson et al., 2012). Furthermore, TGF-b signaling in CNS ECs induces the expression of GPR124 (Anderson et al., 2011; Siqueira et al., 2017) and deletion of GPR124 results in impaired TGF-b signaling in brain ECs (Anderson et al., 2011). Recently, a region-specific mechanism of brain angiogenesis involving TGF-b was also identified (Ma et al., 2017). Here, neural progenitors of the germinal matrix (GM), a unique primordial brain tissue that gives rise to the striatum, regulate their developing vasculature via a GM-specific neurovascular crosstalk. In GM neural progenitors, sphingosin-1-phosphatase signaling via GPCRs regulates the expression of integrin b8, which in turn regulates TGF-b ligand activation, and thus subsequent signaling in brain ECs (Ma et al., 2017). Finally, TGF-b signaling in neurons can also, in a secondary step, regulate CNS vascularization. Indeed, neural deletion of Tgfbr2 hampers EC migration and reduces blood vessel density and branching in the developing telencephalon through altered expression and/or localization of pro- and antiangiogenic factors (Hellbach et al., 2014). 18 Developmental Cell 45, April 9, 2018

The neurite growth-inhibitory membrane protein Nogo-A also plays a role in regulating postnatal angiogenesis in the mouse cortex (Walchli et al., 2013, 2017). Nogo-A is specifically expressed in the neural parenchyma during postnatal angiogenesis and Nogo-A knockout mice display increased number of capillaries and capillary branchpoints. Although the observed phenotype could be secondary to neurodevelopmental defects (Petrinovic et al., 2010), a direct role for Nogo-A on ECs was proposed in vitro (Walchli et al., 2013). Thus, a direct cellular interaction between Nogo-A-expressing neurons and ECs could result in EC cell body and filopodia retraction. Molecular Control of Blood Vessel Patterning during Spinal Cord Vascularization Vascularization of the spinal cord occurs by the formation of the PNVP and the subsequent sprouting of vessels (led by EC tip cells) from the PNVP into the neural tissue (Figure 1B). While the neural tissue, and in particular VEGF derived from it, was known to be crucial for the recruitment and formation of the PNVP (Hogan et al., 2004), the cellular source of VEGF and the specific spatiotemporal process by which the PNVP is formed remained unknown. An elegant study using genetically driven cell ablation approaches in zebrafish recently showed that the PNVP is formed by vessel sprouting from the vertebral arteries and from intersegmental vessels (ISVs), and that this process is regulated by radial glia-derived VEGF, which signals to VEGF receptor-2 (VEGFR-2) in ECs (Matsuoka et al., 2017). The initial sprouting into the spinal cord can be explained by the presence of VEGF and Wnt7a/b expressed by the neural tissue (Daneman et al., 2009; James et al., 2009; Stenman et al., 2008). However, this sprouting does not occur randomly but follows a stereotypic pattern: the first sprouts ingress in between the floor plate and the motor neuron (MN) columns but avoid invading these two regions during a certain developmental time window (Himmels et al., 2017; James et al., 2009) (Figure 1B). Interestingly, as VEGF is expressed in the floor plate and MN columns (Himmels et al., 2017; Ruiz de Almodovar et al., 2011), this stereotyped mode of blood vessel patterning cannot be explained by the sole presence of VEGF (Figure 1B). A gain-of-function study in chick embryos suggested that the stereotypic patterning is achieved by interactions between the growing sprouts and the surrounding neural cells, with most likely VEGF and its antagonists as molecular players (Takahashi et al., 2015). Consistent with this idea, a recent study reported that the VEGF decoy receptor sFlt1 is expressed by MNs (in a NRP1-dependent manner) during the developmental time window where vessels stay out of the MN column soma area (Himmels et al., 2017) (Figure 2A) and that it is responsible for titrating VEGF in MN columns. Changing the VEGF-sFlt1 balance by either overexpressing VEGF in mouse embryos or knocking down sFtl1 in chick embryos results in premature blood vessel ingression into spinal cord MN columns (Himmels et al., 2017) (Figure 2B). sFlt1 is also important for regulating vascular development around this organ (Figure 2C). In zebrafish, Flt1 is detected in spinal cord neurons and in ECs of growing blood vessels (Matsuoka et al., 2016; Wild et al., 2017). One study showed that neuronal-derived sFlt1 is crucial for controlling proper vascularization around the spinal cord as neuronal-specific Flt1 mutants and neuronal-specific sFlt1 knockdown presented a

Developmental Cell

Review A

B

C

D

E

Figure 2. sFlt1 as a Regulator of Spinal Cord Vascularization (A) MN-derived sFlt1 regulates blood vessel patterning in the spinal cord. In the developing spinal cord, MN columns (green) remain avascular until E12.5 in mouse or in Hamburger-Hamilton stage 30 (HH30) chick embryos, despite expressing high levels of VEGF. The avascularization of the MNs in the developing mouse spinal cord, here depicted at E11.5, is ensured via the NRP1-dependent expression of sFlt1 in MNs, which titrates neuronal-derived VEGF and prevents its binding to VEGFRs present in ECs (see inset). (B) Neuronal-specific overexpression of VEGF (inset 1) as well as MN-specific sFlt1 knockdown (inset 2) results in premature vascularization of MN columns due to the higher availability of VEGF for ECs. (C) sFlt1 regulates vascularization around the spinal cord in zebrafish. Neural progenitors (radial glia) regulate sFlt1 levels in ECs. EC-derived sFlt1 and neuronalderived sFlt1 determine the precise patterning of venous vasculature around the developing spinal cord by fine-tuning VEGF levels. Binding of VEGF to neuronalsFlt1 or EC-derived sFlt1 prevents its binding to VEGFRs present in ECs (inset). (D) Neuronal-specific loss of Flt1 or neuronal-specific knockdown of sFlt1 induces venous oversprouting around the spinal cord (black arrows; inset). (E) Neural progenitor (radial glia) ablation leads to venous oversprouting around the spinal cord (black arrows) due to reduced levels of sFlt1 in ECs (inset). The same phenotype is observed in sFlt1 mutants. DA, dorsal aorta; DLAV, dorsal longitudinal anastomotic vessel; EC, endothelial cell; ECM, extracellular matrix; ISV, intersegmental vessel (blue, venous; red, arterial); MN, motor neuron; NP, neural progenitor; PCV, posterior cardinal vein; VEGFR, VEGF receptor.

venous-oversprouting phenotype around the spinal cord (Figure 2D). Premature vessel ingression into the zebrafish spinal cord was further seen when deletion of Flt1 was combined with overexpression of VEGF (Wild et al., 2017) (Figure 2D). Another zebrafish study reported that radial glia (neural progenitor) abla-

tion, as well as sFlt1 mutants, also had a venous-oversprouting phenotype around the spinal cord. Interestingly, they describe a mechanism via which radial glia in the spinal cord control sFlt1 expression levels in ECs, and thus fine-tune the availability of VEGF in ECs (Matsuoka et al., 2016) (Figure 2E). Based on Developmental Cell 45, April 9, 2018 19

Developmental Cell

Review these zebrafish studies, it would be interesting to determine whether similar mechanisms might also be present in mice to control PNVP patterning and the timing of blood vessel ingression into the spinal cord. Together these studies demonstrate that vascular patterning is important for proper spinal cord vascularization and they describe a molecular mechanism by which neuronal progenitors and neurons use VEGF to induce vessel growth. At the same time neurons, or ECs, use the trapping receptor sFlt1 to titrate the availability of VEGF, and thus control vessel sprouting and guidance. They further bring new insights into the cellular mechanisms that the CNS employs to control sFlt1 expression levels in neurons and ECs. Neuronal-Driven Regulation of Retina Vascularization Research using the postnatal mouse retina has provided breakthroughs in the characterization of the different EC types composing a sprouting vessel (tip and stalk ECs) as well as in elucidating the intricate cross-regulation of crucial angiogenic signaling pathways in ECs (e.g., VEGF/VEGFR; Dll4/Notch; BMPs, Wnts, etc.) (Jin et al., 2014; Korn and Augustin, 2015; Potente et al., 2011; Selvam et al., 2017). This model has also been instrumental to characterize how EC metabolism influences angiogenesis (Potente and Carmeliet, 2017). €ller glia, Among the different retinal cell types, astrocytes, Mu microglia, and neurons (retinal ganglion cells [RGCs], amacrine and horizontal neurons) signal to each other and to ECs in order to control developmental angiogenesis. EC-to-EC communication is also crucial. In this section we mainly discuss the involvement of retinal neurons in retina vascularization. For an entire overview of retina angiogenesis, we refer to the recent review by Selvam et al. (2017). As in other parts of the CNS, VEGF plays a crucial role in retinal angiogenesis. The major source of VEGF in the retina is glia cells €ller glia) (Gerhardt et al., 2003; West et al., (astrocytes and Mu 2005), where VEGF expression is driven by hypoxia (Selvam et al., 2017). The number of retinal astrocytes is controlled by RGC-derived PDGF-A that signals to PDGFRa expressed by astrocytes (Fruttiger et al., 1996; West et al., 2005). Overexpression of neural-PDGF-A causes a denser astrocytic network that concomitantly results in an increased vessel density (Fruttiger et al., 1996). Maturation of the astrocytic network is in return controlled by the endothelium via a negative feedback loop where astrocytes reduce proliferation and VEGF expression upon the contact with blood vessels and the supply of oxygen (Duan et al., 2014; Kubota et al., 2008; Sakimoto et al., 2012; Uemura et al., 2006; West et al., 2005). Interestingly, selectively €ller glia does not result in deleting VEGF in either astrocytes or Mu significant vascular phenotypes during development (Bai et al., 2009; Weidemann et al., 2010), suggesting that other retinal cell types can also produce VEGF (and other angiogenic factors). In this sense, conditional ablation of RGCs completely abolishes developmental retina angiogenesis, even though a retinal astrocytic network is still present (Sapieha et al., 2008), indicating that the neuronal compartment is crucial for proper retina vascularization. Expression of VEGF and Angiopoietin-1 is also regulated in RGCs during retina vascularization via a mechanism involving the succinate receptor GPR91 and the coagulation factor II receptor-like 1 (F2rl1), expressed in RGCs (Joyal et al., 2014; 20 Developmental Cell 45, April 9, 2018

Sapieha et al., 2008). Consistently, knockdown of GPR91 or F2rl1 reduces retinal developmental angiogenesis (Joyal et al., 2014; Sapieha et al., 2008). RGCs also express VEGFR2 (Okabe et al., 2014), and its presence in these neurons regulates retinal angiogenesis by titrating the levels of VEGF, and thus controlling the direction of newly formed vessel sprouts (Okabe et al., 2014). Hence, when compared with the spinal cord (where neuronalderived sFlt1 titrates VEGF), it seems that different neurons use different VEGF receptor-mediated mechanisms to titrate VEGF levels and control blood vessel patterning in the CNS. Interactions between neurons from the inner nuclear layer (INL) of the retina and capillaries from the intermediate and deep vascular plexus have been described (Usui et al., 2015). In particular, amacrine and horizontal neurons express VEGF in an HIF1a-dependent manner, and this source of VEGF is required for vessel sprouting and branching in the deep and intermediate plexus (Usui et al., 2015). A crosstalk between VEGF/VEGFR2 and Slit2/Robo1/Robo2 signaling pathways in ECs was recently discovered (Rama et al., 2015). Here, Slit2, expressed by INL neurons and by ECs, promotes retinal angiogenesis through Robo1/Robo2 on ECs. Slit2 regulates EC migration via RAC1 activation and lamellipodia formation. Notably, Robo1 and Robo2 also seem to be required for VEGF-induced RAC1 activation (Rama et al., 2015), indicating a crosstalk between both signaling pathways. Interestingly, while co-stimulation of ECs with Slit2 and VEGF promoted EC migration, pre-treatment of ECs with Slit2 resulted in VEGFR2 internalization and inhibited VEGF signaling (Rama et al., 2015), suggesting that depending on the distribution of Slit2, with respect to VEGF, its signaling could induce one or the other effect on ECs. RGC-derived Sema3E signaling to PlexinD1 in ECs also regulates retina angiogenesis (Fukushima et al., 2011; Kim et al., 2011), as lack of Sema3E or PlexinD1 leads to an uneven growing vascular front and a less branched vascular network (Kim et al., 2011). PlexinD1 expression in ECs is dynamically regulated by VEGF at the front of actively sprouting blood vessels and Sema3E/Plexin-D1 signaling in ECs fine-tunes VEGF’s activity in a negative feedback loop by suppressing Dll4 expression in tip cells (Kim et al., 2011). Neuronal Activity Modulates Vascular Development While it was well known that neuronal activity is important for controlling cerebral blood flow, it is only recently that a link between neuronal activity and cerebrovascular patterning during development was described. The use of different paradigms of sensory deprivation in mouse pups revealed that a reduction of the sensory input led to decreased EC proliferation, vascular density, and branching in the barrel cortex (Lacoste et al., 2014). Enhancement of neuronal activity by increasing sensory inputs just had the opposite effect (Lacoste et al., 2014). While Lacoste et al. (2014) described reduced blood vessel density and branching when reducing the sensory input during postnatal development, another study did not observe any difference in vascular development when comparing basal conditions and conditions of reduced sensory input (Whiteus et al., 2014). In contrast, this study showed that excessive sensory stimulation as well as chemically induced seizures led to a reduction in EC proliferation and vessel sprouting, which in turn affected the

Developmental Cell

Review cerebrovascular patterning during development. The vasculature was not affected in adult mice after altering neural activity (Whiteus et al., 2014). Although these studies report contradictory findings, part of this contradiction might be explained by the distinct methodology used for image analysis of the cerebral vasculature, as indicated in Lacoste et al. (2014). Still, they indicate that neural activity regulates angiogenesis in the developing brain and that vessel branching and maturation does not only rely on EC/pericyte intrinsic programs but also on stimuli received from the environment. Thus, further investigations are needed to elucidate the mechanisms of how local neuronal activity affects vessel patterning during development and whether neuronal activity could be used to modulate angiogenesis in pathological conditions. Vascular Control of Neurogenesis during CNS Development During CNS development and in certain regions of the adult brain, neural stem cells (NSCs) and neural progenitor cells (NPCs) reside in neurogenic niches, with blood vessels being a crucial component of these niches (Bjornsson et al., 2015). In adult neurogenic niches, the vasculature is required not only for delivery of oxygen and nutrients but also for trophic support of the neuronal compartment (Licht and Keshet, 2015; Ramasamy et al., 2015). However, little is known about the influence that blood vessels have on neurogenesis during development. Neurogenesis begins in germinal zones around E9 to E10 of mouse development (Kriegstein and Alvarez-Buylla, 2009). The timing of CNS angiogenesis parallels the gradient of neurogenesis and tissue expansion in the forebrain (Vasudevan et al., 2008), hindbrain (Tata et al., 2016; Ulrich et al., 2011), and spinal cord (Himmels et al., 2017; Hogan et al., 2004; Takahashi et al., 2015), consistent with the fact that neural tissue expansion requires proper oxygenation and nutrient delivery to meet the metabolic needs of the growing populations (Knobloch and Jessberger, 2017). Proper blood vessel growth and patterning during CNS development seem to be crucial for defining the location of neurogenic niches as a perturbation of blood vessel growth in organotypic cultures of embryonic brains results in the mispositioning of NPCs to non-neurogenic regions (Javaherian and Kriegstein, 2009; Li et al., 2013). In this respect, earlier studies showed that NPCs reside in close association to blood vessels in the embryonic and postnatal brain and that the developing vasculature provides a proliferative microenvironment for NPCs (Javaherian and Kriegstein, 2009; Nie et al., 2010). Interestingly, while NPCs from the ventral telencephalon (giving rise to inhibitory neurons) seem to require the association to blood vessels, NPCs from the dorsal region (giving rise to excitatory neurons) do not (Tan et al., 2016). Although both NPC populations are initially anchored to the pial basement membrane, from E14.5 ventral telencephalic NPCs shift their anchorage to periventricular blood vessels via a mechanism involving integrin b1 expressed by their basal processes and basement membrane laminin-enriched sheet covering blood vessels (Tan et al., 2016). This interaction is necessary to promote NPC division and neocortical interneuron neurogenesis. Neurogenic niches are hypoxic, and this condition is required for NSC proliferation (Mohyeldin et al., 2010). In the embryonic forebrain, a close spatiotemporal relationship between blood

vessel formation and NSC behavior exists, and the relief of hypoxia by angiogenesis promotes NSC differentiation (Lange et al., 2016). When blood vessel growth is blocked during CNS development, cortical NSC expansion is increased at the expense of NSC differentiation. This switch is controlled by HIF1a in NSCs (Lange et al., 2016). Oxygen tension during cortical neurogenesis is also essential for establishing neural progenitor identities (Wa€ hr et al., 2015). Similarly, developmental neurogenesis is genfu influenced by the vascular niche in the embryonic hindbrain (Tata et al., 2016). EC-specific deletion of NRP1 results in reduced angiogenesis in the hindbrain and concomitantly in premature neuronal differentiation at the expense of reduced NPC self-renewal (Tata et al., 2016). However, EC-specific NRP1 knockout hindbrains show increased levels of HIF1a and VEGF (hypoxia markers) and restoration of tissue oxygenation does not rescue the phenotype (Tata et al., 2016). Thus, in the hindbrain, it seems that tissue oxygenation is not sufficient for proper neurogenesis but that, together with proper tissue oxygenation, EC-derived factors (which would be absent in EC-specific NRP1 knockout hindbrains) are also required. In summary, the above-mentioned studies highlight the central role of the vascular niche for developmental neurogenesis (Table 2). While the role of hypoxia is relatively well understood as a key component of neurogenic niches, the identification of blood vessel-derived signals influencing NSCs/NPCs during embryonic development requires further investigation. Vascular-Derived Signals that Influence NSCs While blood vessels in the adult subventricular zone (SVZ) neurogenic niche actively secrete angiocrine cues to influence NSC behavior (Licht and Keshet, 2015), molecular factors influencing NSC behavior during CNS development have been less characterized. Below we describe the identified cellular mechanisms and molecular cues to date and provide an overview in Table 2. Gene expression profile of embryonic and neonatal brain ECs showed a distinctive and highly dynamic gene temporal expression pattern and identified genes encoding brain endothelium specifically, as well as several other molecules differentially expressed in CNS ECs (Daneman et al., 2010; Hupe et al., 2017). Interestingly, many of the enriched molecules in postnatal brain ECs, such as CXCL12, semaphorins, pleiotrophin (Ptn), and Wnt signaling pathway components, have been implicated in regulating NSC behavior (Bjornsson et al., 2015; Daneman et al., 2010; Licht and Keshet, 2015), suggesting that a cellular source for those factors at the developing neurogenic niche might be the endothelium. In support of this, several in vitro studies showed that EC-derived factors promote neurogenesis in the embryonic cortex, spinal cord, and postnatal brain. Specifically, embryonic and postnatal NPCs respond differently to diffusible signals of ECs than to direct cell-cell interactions in co-culture systems. While diffusible cues are thought to maintain NSCs/NPCs, inhibiting or delaying their differentiation (Gama Sosa et al., 2007; Li et al., 2006; Lowry et al., 2008; Shen et al., 2004; Vissapragada et al., 2014; Weidenfeller et al., 2007), direct cell-cell contact promotes neural differentiation (Gama Sosa et al., 2007; Guo et al., 2008; Plane et al., 2010). Recently, EC-derived Notch-ligand Jagged and EC-derived EphrinB2 were identified to be critical for maintaining neonatal SVZ NSCs in a quiescent state, jointly inhibiting differentiation (Ottone Developmental Cell 45, April 9, 2018 21

22 Developmental Cell 45, April 9, 2018

Table 2. Cellular and Molecular Mechanisms Involved in EC to Neural Communication during CNS Development CNS Cell Type

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines Used/In Vitro Studies

Comments

Reference

ECs

not defined

NPCs

mouse/hindbrain (E11.5)

Nrp1 / Tie2-cre x Nrp1 flox/flox

hindbrain vascularization sustains NPC proliferation and regulates NPC differentiation

(Tata et al., 2016)

ECs

not defined

NSCs/NPCs

mouse/brain (telencephalon) (E13.5)

Gpr124 / PDGFRb-ncre x Gpr124 flox/flox Emx1tm1-cre x HIF-1aflox/flox

vascularization and relief of tissue hypoxia by HIF1a destabilization in NSCs/ NPCs induces neural differentiation

(Lange et al., 2016)

ECs

VEGF?

NPCs/postmitotic neurons

mouse/brain (cortex) (E17)

Tie2-cre x Vegf flox/flox

EC-derived VEGF is required for proliferation of NPCs and migration of neurons, regulating cortical lamination

(Li et al., 2013)

ECs

EphrinB2/Jagged

NSCs

mouse/brain (SVZ) (P6–P10)

Cdh5(PAC)-creERT2 x Jagflox/flox Cdh5(PAC)-creERT2 x Efnb2flox/flox

EC-derived EphrinB2 sustains NSC quiescence via Eph receptors; EC-derived Jagged induces type-B stem cell identity via Notch signaling in NSCs

(Ottone et al., 2014)

ECs

not defined

NSCs

mouse/spinal cord (E8–E9)

in vitro study

EC-NSC co-culture induces NSC proliferation

(Lowry et al., 2008)

ECs

VEGF

NSCs

rat/brain (SVZ) (P3)

in vitro study

EC-derived VEGF induces NSC proliferation and differentiation

(Sun et al., 2010)

ECs

not defined

NSCs

mouse/brain (cortex) (E10–E11)

in vitro study

EC-soluble factors induce embryonic NSC selfrenewal, neuron specification, and inhibit differentiation

(Shen et al., 2004)

ECs

not defined

NSCs

mouse/brain (E13.5–E16)

in vitro study

EC-soluble factors maintain embryonic NSC stemness

(Gama Sosa et al., 2007; Vissapragada et al., 2014)

ECs

not defined

postmitotic neurons

mouse/brain (cortex) (E16–E18)

in vitro study

EC-soluble factors promote survival and maturation of neurons

(Wu et al., 2016; Wu et al., 2017)

ECs

laminin

NSCs

mouse/brain (SVZ) (P1–P3)

in vitro study

a6b1 integrin in NSCs establishes direct contact with ECs via laminin, inducing NSC proliferation and stemness

(Rosa et al., 2016)

(Continued on next page)

Developmental Cell

Vascular Signal

Review

Vascular Cell Type

Developmental Cell 45, April 9, 2018 23

Vascular Cell Type

Vascular Signal

CNS Cell Type

Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines Used/In Vitro Studies

Comments

Reference

ECs

Wnt3a

NSCs

rat/spinal cord (P5–P7)

in vitro study

co-culture induces NSC proliferation and differentiation via ECderived Wnt3a

(Du et al., 2016)

ECs

not defined

NSCs

mouse/brain (SVZ) (P15)

in vitro study

EC-NSC soluble factors promote neural stem/ progenitor-like phenotype and direct EC-NSC contact induces differentiation

(Plane et al., 2010)

Vasculature

not defined

optic nerve neurons

mouse/optic pathway (E11.5–E15.5)

Brn3-cre/+ x Nrp1flox/Tie2-cre x Nrp1flox/Vegfa120/120 Vegfa188/188

vascular patterning is crucial for axonal organization in the optical pathway

(Erskine et al., 2017)

Vasculature

laminin

NPCs

mouse/brain (telencephalon) (E12.5)

Nkx2.1-cre x Itgb1flox/flox

ventral NSCs anchor to periventricular blood vessels, which regulates proliferation and differentiation; deletion of integrin b1 disrupts blood vessel-NPC association

(Tan et al., 2016)

ECs

GABA

GABAergic neurons

mouse/brain (telencephalon) (E13)

WT mice in vitro studies

ECs produce GABA, which in turn regulates GABAergic neuron tangential migration; GABRA2+ pial ECs instruct superficial neuron migration; GABRB3+ periventricular ECs instruct deep neurons migration

(Won et al., 2013)

Vasculature involved (cell type not defined)

not defined

GABAergic neurons

mouse/brain (telencephalon) (E12.5–P1)

Camklla-tTA x pTETsVEGFR1

angiogenesis impairment leads to neural apoptosis and impaired GABAergic neuron migration

(Licht et al., 2015)

Vasculature involved (cell type not defined)

not defined

neuroblasts

mouse/brain (white matter) (P4–P8–P12)

WT mice Reporter mouse line (5HT3-EGFP)

vasculature serves as a scaffold for radial migration of neuroblasts through corpus callosum into lower layers of cortex

(Le Magueresse et al., 2012)

ECs

not defined

neuroblasts

mouse/brain (RMS) (P3–P21)

WT mice

blood vessel remodeling by astrocyte-secreted VEGF allows efficient neuroblast tangential migration through RMS

(Bozoyan et al., 2012)

(Continued on next page)

Developmental Cell

Continued

Review

Table 2.

24 Developmental Cell 45, April 9, 2018

Table 2.

Continued Organism/CNS Region (Developmental Stage)

Genetic Modified Mouse Lines Used/In Vitro Studies

Comments

Reference

young neurons

human/brain (RMS) (postnatal)

not applicable

blood vessels serve as a scaffold for tangential migration of human DCX+ young neurons during embryonic development

(Paredes et al., 2016)

CXCL12

OLPs

mouse/brain (telencephalon and spinal cord) (E14)

Gpr124 / , PDGFRb / CXCR4 / Cdh5-cre x Gpr124 flox/flox Olig2-cre x APC flox/flox

blood vessels serve as a scaffold for OLP migration. CXCR4+ OLPs are attracted to CXCL12 expressing blood vessels. This association sustains migration and inhibits oligodendrocyte maturation

(Tsai et al., 2016)

ECs

not defined

OLPs

white matter P1–P2

in vitro study

EC-derived soluble factors support OLP survival and proliferation

(Arai and Lo, 2009)

ECs

VEGF

OLPs

white matter (P1–P2)

in vitro study

EC-derived VEGF promotes OLP migration at least in part via Flk1 expressed in OLPs

(Hayakawa et al., 2011; Hayakawa et al., 2012)

Pericytes/Meninges

TGF-b-BMP

OLPs

mouse/brain (cortex) (E15.5–E17.5)

PDGFR-b-cre x FoxC1 flox/flox PDGFR-b-cre x TGF-b flox/flox

pericyte-derived BMP and TGF-b repel OLPs from ventral forebrain favoring dorsal migration into cortex via BMP receptor Ia during corticogenesis

(Choe et al., 2014)

Vascular Cell Type

Vascular Signal

CNS Cell Type

Vasculature involved (cell type not defined)

not defined

Vasculature ECs

Developmental Cell

Review

Developmental Cell

Review et al., 2014). Similarly, an in vitro study showed that EC-derived laminin signals via a6b1-expressing NSCs to induce proliferation and to preserve stem cell identity (Rosa et al., 2016). Vascular-toNPC communication is also required for trophic support of NPCderived neural and glial progeny as in vitro studies have shown that EC-soluble factors enhance postmitotic neuronal survival (Dugas et al., 2008; Wu et al., 2016) and induce neuronal maturation (Dugas et al., 2008; Wu et al., 2017). Although the main sources of VEGF during CNS development are NSCs and their progeny (Himmels et al., 2017; James et al., 2009; Okabe et al., 2014; Raab et al., 2004), developing brain ECs also express VEGF (Li et al., 2013; Sun et al., 2010; Virgintino et al., 2003) and the VEGF present in brain EC-conditioned medium potentiates cortical neuron morphological and electrophysiological development via VEGFR2/p38 MAPK signaling (Wu et al., 2017). In vivo, VEGF EC-specific knockout embryos present defects in NPC proliferation, differentiation, and lamination (Li et al., 2013), suggesting that EC-derived VEGF controls corticogenesis. However, as VEGF EC-specific knockout embryos also present impaired angiogenesis (Li et al., 2013) and VEGF-depleted ECs have a different gene expression profile than wild-type ECs (Sun et al., 2010), it remains to be determined whether some of the observed effects in corticogenesis could appear as a consequence of the latter differences. Although Wnt ligands and other modulators of Wnt signaling are secreted by ECs during development, there is no in vivo evidence for an EC-to-NSC communication involving Wnts that regulate neurogenesis. However, a recent co-culture study of embryonic bone marrow progenitor ECs with rat newborn-spinal cord NSCs shows that EC-derived Wnt3a is necessary for NSC proliferation and differentiation (Du et al., 2016). Role of the CNS Vasculature as a Guidance or Physical Track for Newborn Neurons and Axon Projections During CNS developmental and adult neurogenesis, neuroblasts, neurons, OLPs, and astrocytes migrate from germinal zones toward their final destinations using specific molecular cues and tracks provided by cellular substrates that build their migration pathways. Classically, radial glia and neuronal axons have been shown to act as scaffolds to support neural precursor locomotion (Saghatelyan, 2009). Remarkably, during CNS development and adulthood, blood vessels are also operating as cellular substrates for neuroblast (Snapyan et al., 2009) and neuronal migration (Won et al., 2013). Notably, vasculaturemediated young neuron tangential migration from germinal zones to cortical areas is also conserved in the human infant brain (Paredes et al., 2016). In the adult SVZ neurogenic niche, chains of neuroblasts migrate through the rostral migratory stream (RMS) from the posterior SVZ to the anterior olfactory bulb using blood vessels as a physical and trophic substrate (Licht and Keshet, 2015). The RMS starts being vascularized by E14.5 and continues during postnatal development (Colin-Castela´n et al., 2011). During early developmental stages, in a process regulated by astrocytederived VEGF, blood vessels orient mostly radially, and between P14 and P21 align along the RMS to allow efficient tangential neuroblast migration in adulthood (Bozoyan et al., 2012). In addition to the migration of neurons along blood vessels of the RMS, neurons of the SVZ also migrate radially along the vasculature to

lower cortical layers during the first four postnatal weeks (Le Magueresse et al., 2012). Here, postnatal astrocytes also associate with blood vessels and orchestrate the organization of the vascular scaffold for neuroblast migration in the corpus callosum, suggesting equivalent migratory mechanisms within different regions of the developing postnatal brain (Bozoyan et al., 2012; Le Magueresse et al., 2012) (Figure 3A). During early embryonic cortical development, the majority of neurons born in the ganglionic eminence migrate through areas free of axons and only a minor population uses intermediate zone axons for tangential migration (Marin and Rubenstein, 2003), suggesting that the growing vasculature could act as a scaffold for these navigating neurons. Consistent with this hypothesis, impairment of the developing vasculature by trapping VEGF interrupts migration of E13 ganglionic eminence-born GABAergic neurons toward superficial cortical layers (Licht et al., 2015). In addition, the different spatial migratory routes (deep and superficial streams) that GABAergic neurons from the embryonic telencephalon take toward the cortex seem to depend on a regionalized vasculature, to which GABAergic neurons associate closely (Won et al., 2013) (Figure 3B). Periventricular ECs have a different gene expression profile and intrinsic program compared with pial ECs. This, and the fact that deep neurons respond to low GABA levels secreted by periventricular ECs, and superficial neurons to high GABA levels secreted by pial ECs, results in two well-defined EC populations that display distinct chemoattractive activity to deep or superficial GABAergic neurons (Won et al., 2013). Interestingly, the developing vasculature also plays a role in shaping axon projections of RGCs at the optic chiasm. Manipulation of vascular patterning by in vivo EC-specific deletion of NRP1, or by the sole expression of a single VEGF isoform (VEGF188/188 or VEGF121/121 mice), results in an abnormal eye and optic chiasm-surrounding vasculature that affects optic tract organization. As blood vessels did not repel axons in vitro and in vivo, it was concluded that these aberrant blood vessels present a physical obstacle for correct RGC axon growth, which results in axonal exclusion zones at the midline and in the optic tract (Erskine et al., 2017). Similarly, a mouse line that has increased blood vessel density and deregulated patterning in MN columns during spinal cord development, due to the overexpression of VEGF in neurons, displayed an erroneous distribution of columnar MN somas and their axons exit the CNS in an aberrant defasciculated manner (Himmels et al., 2017). Thus, correct vascular growth and patterning during CNS development is critical for the precise orchestration of neurodevelopmental processes such as axon projection and cell soma arrangements. Regulation of Oligodendrocyte Migration by Blood Vessels Oligodendrocytes (OLs), the myelinating cells of the CNS, also use blood vessels for their migration during CNS development. Mature OLs arise from a restricted OLP population specified around E12.5 from the medial ganglionic eminence in the telencephalon and the MN progenitor domain in the spinal cord. OLPs migrate from their birthplace to homogeneously populate the entire developing CNS (Kessaris et al., 2006). Interestingly, a recent study shows that embryonic OLPs intimately associate with the vasculature to promote their migration and Developmental Cell 45, April 9, 2018 25

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Review A

B

Figure 3. Vascular Control of Neuronal Migration (A) Vascular-guided migration of neurons in the postnatal brain. During postnatal development, new neurons are continuously generated in the SVZ and neuroblasts migrate radially toward the cortex (CX) through the corpus callosum (CC; inset, radial migration) and tangentially through the RMS toward the olfactory bulb (OB, inset, tangential migration). Both migration pathways use blood vessels (BV) as scaffolds. Vasculature formation and structural organization in the RMS is orchestrated by astrocytes (A, pink) secreting VEGF (inset, tangential migration). CB, cerebellum; HC, hippocampus; LV, lateral ventricle; N, neuron/neuroblast; RMS, rostral migratory stream; SVZ, subventricular zone. (B) Vascular-guided migration of neurons in the embryonic brain. Superficial and deep GABAergic neurons born in the medial ganglionic eminence (MGE; around E12.5–E13.5) migrate dorsally associated to the vasculature toward the neocortex, avoiding the striatum (Str). Superficial neurons respond to high levels of GABA secreted by pial ECs (inset, superficial neurons). Deep neurons respond to lower levels of GABA secreted by periventricular ECs (inset, deep neurons).

concomitantly prevent maturation (Tsai et al., 2016). This association occurs via the following molecular crosstalk: OLP autocrine-derived Wnt signaling induces the expression of the chemokine receptor CXCR4 in OLPs (Figure 4). EC-derived CXCL12 attracts CXCR4-expressing OLPs to the vasculature and allows their migration, preventing OL maturation (Figure 4). Close contact of telencephalic NG2+ OLPs with blood vessels in the embryonic cortex is also important for branching and connectivity between blood vessels, as well as for vascular network refinement (Minocha et al., 2015). As soon as OLPs reach their final destination, Wnt signaling is downregulated in OLPs and 26 Developmental Cell 45, April 9, 2018

OLPs mature by detaching from the scaffolding vasculature (Figure 4). Pericyte-deficient embryos have no OLP migration impairment at embryonic developmental stages (Tsai et al., 2016), indicating that pericytes are not required for the association to blood vessels and the migration along them. Still, pericyte- and meningeal-secreted factors (BMPs and TGF-b) seem to be required during embryonic development for repelling OLPs from their ventral birthplaces, and thus promoting their migration toward the cortex (Choe et al., 2014). Remarkably, EC-secreted factors have been shown to increase OLP (Arai and Lo, 2009) and OL (Plane et al., 2010)

Developmental Cell

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B

Figure 4. Crosstalk between Blood Vessels and OLPs during Development (A) During development OLPs migrate dorsally (arrows) from their birthplace in the ventral spinal cord and ventral forebrain to homogeneously populate the CNS. (B) OLPs are guided by the vasculature. OLPs are specified around E12.0 from NPCs and immediately associate to blood vessels. OLP-secreted Wnt acts in an autocrine manner to induce expression of CXCR4 (inset 1). In turn, blood vessels secrete CXCL12 (CXCR4 ligand) to attract OLPs and sustain their undifferentiated state (inset 1). Maturation of oligodendrocytes (OLs) is accompanied by downregulation of Wnt expression, as well as CXCR4, in OLPs and concomitant OLP detachment from the vasculature (inset 2). At P0, physiological hypoxia triggers stabilization of HIF1/2a (Hifa) in OLPs, which induces Wnt7a/b expression in OLPs to signal in an autocrine manner and arrest their maturation. Simultaneously, Wnt7a/b acts on ECs to promote angiogenesis (inset 3). As oxygen tension increases due to formation of new blood vessels, HIF1/2a is destabilized yielding to a reduction of Wnt7a/b production (inset 4) and resulting in the final maturation of OLPs, which detach from the vasculature.

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Review numbers in vitro. In this respect, EC-derived VEGF affected white matter OLP migratory behavior via VEGFR2 in cell culture (Hayakawa et al., 2011, 2012), but whether this also occurs in vivo remains to be determined. Altogether, these studies suggest that a differential communication/association of embryonic OLPs and postnatal OLPs with the vasculature exists and that this association could depend on OLP functional demands. They also indicate that the role of the vasculature is more complex than only acting as a migration substrate for OLPs. Conclusions and Future Perspectives Despite the importance of a neurovascular crosstalk, we are just beginning to understand the underlying mechanisms of this bidirectional communication and their implications. As discussed in this review, ECs and neural cells have evolved unique mechanisms and ways to communicate with each other within the CNS in order to guarantee its proper development and maintenance. Secretion of molecular cues (e.g., VEGF and sFlt1, Wnts, TGF-b, etc.) from the neural tissue, as well as the direct cellcell contact of different neural cell types with ECs, regulates embryonic and postnatal CNS angiogenesis by influencing the EC proliferation, differentiation, and migration. Interestingly, neural activity can also modulate vascular development, a finding that opens a completely new perspective in neurovascular communication. However, many important questions remain unanswered. (1) As indicated by several studies (Himmels et al., 2017; Ma et al., 2017; Vasudevan et al., 2008), it seems that region-specific mechanisms of CNS angiogenesis are important for controlling its proper vascularization. How is vascularization of other CNS regions then controlled and what are the cellular and molecular mechanisms? (2) How is the patterning of the invading sprouts regulated? (3) Is VEGF signaling in ECs modulated via other signaling pathways (e.g., Wnt, TGF-b, Nogo-A)? In this sense, VEGF induces angiogenesis via the activation of the Hippo pathway effectors YAP/TAZ (Kim et al., 2017; Wang et al., 2017), which are also regulated by Wnt and TGF-b signaling (Hansen et al., 2015), so, could there be a molecular interaction at this level? (4) To what extent is neuronal activity required for controlling developmental angiogenesis in different CNS regions? (5) Do the principal mechanisms of this bidirectional communication in physiological conditions become re-activated or de-regulated in pathological situations? Development of the nervous tissue highly depends on the developing vasculature. Indeed, in the developing CNS, the nascent vasculature regulates NSC behavior and embryonic neurogenesis (Lange et al., 2016; Li et al., 2013; Tata et al., 2016) by providing oxygen and specific signaling cues to the expanding nervous tissue in order to drive neural proliferation and differentiation (Lange et al., 2016; Tan et al., 2016). Migration of NPCs, GABAergic neurons, and OLPs also depends on blood vessels (Bozoyan et al., 2012; Tsai et al., 2016; Won et al., 2013). However, the nature of the EC-derived cues required for these processes remains elusive. Developing CNS ECs show high heterogeneity in their gene expression profile (Hupe et al., 2017), not only compared with other organ ECs, but also within the CNS (Won et al., 2013). 28 Developmental Cell 45, April 9, 2018

Future studies should address this EC heterogeneity and the distinctive properties of ECs residing in specific regions of the CNS. The outcome could explain, for example, the regional and differential influence of ECs in NSC behavior and their progeny. To date, the contribution of mural cells, namely vasculature pericytes and vascular smooth muscle cells, to the neurovascular niche remains elusive. Thus, to understand the overall functional relevance of this crosstalk in the developing CNS, their functionality in CNS neuro- and vascular development should be studied. Finally, as a number of brain disorders are accompanied by vascular dysfunction (e.g., BBB disruption, edema formation, vascular hemorrhages, cerebral palsy, etc.), it is important to understand the precise mechanisms of the communication between neurons, vessels, and other cell types of the neurovascular unit in order to better understand neuronal disorders and find new therapeutic treatments. ACKNOWLEDGMENTS We apologize for those whose work we could not cite due to space constrictions. We thank all Ruiz de Almodo´var’s lab members for their scientific input. C.R.A. is supported by ERC (ERC-StG-311367), DFG FOR-2325, DFG SFB873, and the Schram Foundation. REFERENCES Adams, R.H., and Eichmann, A. (2010). Axon guidance molecules in vascular patterning. Cold Spring Harb. Perspect. Biol. 2, a001875. Allinson, K.R., Lee, H.S., Fruttiger, M., McCarty, J.H., and Arthur, H.M. (2012). Endothelial expression of TGFbeta type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system. PLoS One 7, e39336. Anderson, K.D., Pan, L., Yang, X.M., Hughes, V.C., Walls, J.R., Dominguez, M.G., Simmons, M.V., Burfeind, P., Xue, Y., Wei, Y., et al. (2011). Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor. Proc. Natl. Acad. Sci. USA 108, 2807–2812. Arai, K., and Lo, E.H. (2009). An oligovascular niche: cerebral endothelial cells promote the survival and proliferation of oligodendrocyte precursor cells. J. Neurosci. 29, 4351–4355. Arnold, T.D., Niaudet, C., Pang, M.F., Siegenthaler, J., Gaengel, K., Jung, B., Ferrero, G.M., Mukouyama, Y.S., Fuxe, J., Akhurst, R., et al. (2014). Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking alphaVbeta8-TGFbeta signaling in the brain. Development 141, 4489–4499. Augustin, H.G., and Koh, G.Y. (2017). Organotypic vasculature: from descriptive heterogeneity to functional pathophysiology. Science 357, eeal2379. Bai, Y., Ma, J.X., Guo, J., Wang, J., Zhu, M., Chen, Y., and Le, Y.Z. (2009). Muller cell-derived VEGF is a significant contributor to retinal neovascularization. J. Pathol. 219, 446–454. Bjornsson, C.S., Apostolopoulou, M., Tian, Y., and Temple, S. (2015). It takes a village: constructing the neurogenic niche. Dev. Cell 32, 435–446. Bonney, S., Harrison-Uy, S., Mishra, S., MacPherson, A.M., Choe, Y., Li, D., Jaminet, S.C., Fruttiger, M., Pleasure, S.J., and Siegenthaler, J.A. (2016). Diverse functions of retinoic acid in brain vascular development. J. Neurosci. 36, 7786–7801. Bozoyan, L., Khlghatyan, J., and Saghatelyan, A. (2012). Astrocytes control the development of the migration-promoting vasculature scaffold in the postnatal brain via VEGF signaling. J. Neurosci. 32, 1687–1704. Cho, C., Smallwood, P.M., and Nathans, J. (2017). Reck and Gpr124 are essential receptor cofactors for Wnt7a/Wnt7b-specific signaling in mammalian CNS angiogenesis and blood-brain barrier regulation. Neuron 95, 1221–1225.

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