Article

A brain-specific angiogenic mechanism
enabled by tip cell specialization
https://doi.org/10.1038/s41586-024-07283-6
Received: 2 June 2023
Accepted: 7 March 2024

Giel Schevenels1, Pauline Cabochette1, Michelle America1, Arnaud Vandenborne1,
Line De Grande1, Stefan Guenther2, Liqun He3, Marc Dieu4, Basile Christou1,
Marjorie Vermeersch5, Raoul F. V. Germano1, David Perez-Morga1,5, Patricia Renard4,
Maud Martin1, Michael Vanlandewijck3,6, Christer Betsholtz3,6 & Benoit Vanhollebeke1 ✉Published online: 3 April 2024
Open access
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Vertebrate organs require locally adapted blood vessels1,2. The gain of suchorganotypic vessel specializations is often deemed to be molecularly unrelated
to the process of organ vascularization. Here, opposing this model, we reveal amolecular mechanism for brain-specific angiogenesis that operates under thecontrol of Wnt7a/b ligands—well-known blood–brain barrier maturation signals3–5.
The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which
we find is enriched in brain endothelial cells. CRISPR–Cas9 mutagenesis in zebrafish
reveals that this poorly characterized glycosylphosphatidylinositol-anchoredmatrix metalloproteinase is selectively required in endothelial tip cells to enable
their initial migration across the pial basement membrane lining the brain surface.
Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal
fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of
the central helical part of the heterotrimer. After genetic interference with the pial
basement membrane composition, the Wnt–β-catenin-dependent organotypic
control of brain angiogenesis is lost, resulting in properly patterned, yet blood–
brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis
mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate
how organs, by imposing local constraints on angiogenic tip cells, can select vessels
matching their distinctive physiological requirements.

In vertebrates growing beyond the limit of oxygen diffusion, a vascularsystem branches through the body to supply nutrients and oxygen,remove cellular waste products, allow immune cell transport and support coagulation1,6. Contrasting with the apparent uniformity of theirancestral functions, blood vessels exhibit considerable phenotypic
heterogeneity. In particular, the inner lining of blood vessels is madeof highly malleable endothelial cells (ECs) that engage in a varietyof two-way communications with local microenvironments1,2,7. By
adopting organ-specific structural and molecular profiles, ECs haveessential roles in organ development, metabolism, regeneration andrepair. As angiogenesis is almost invariably initiated by an environmental trigger common to all expanding organs, that is, the shortageof oxygen, the cellular and molecular logic of blood vessel branching
through sprouting angiogenesis and organotypic endothelial adaptations are generally viewed as distinct events, regulated by independent
molecular machineries.
The central nervous system (CNS) offers a notable exception tothis rule. From early developmental stages onwards, its functional
complexity requires isolation and protection from peripheral chemical variations and toxins. Embryonic cerebral ECs therefore mature
into a neuroprotective blood–brain barrier (BBB) phenotype, anarchetypical example of EC specialization, that is initiated by endothelial Wnt–β-catenin signalling3–5. In response to Wnt ligands, CNS ECs
reduce paracellular permeability through tight junctions, repress
transcytosis, express numerous solute transporters such as GLUT1
and recruit pericytes. While, as in any other organ, brain angiogenesis
is VEGF-dependent, the BBB-inductive Wnt–β-catenin signalling pathway is also required for brain angiogenesis, thereby making a direct,
yet enigmatic, link between organ vascularization and organotypicendothelial specialization.
Through this coupling mechanism, no leaky blood vessels penetrate
the delicate neuroepithelium, accounting for an efficient endothelial
quality-control mechanism. However, how Wnt signalling impinges
on the angiogenic cascade selectively in the brain and, thereby, moregenerally, how organs can ensure that their perfusing vessels match
their specific metabolic requirements remain to be determined.

Brain-specific angiogenesis control
To investigate the organotypic control of brain angiogenesis byendothelial Wnt–β-catenin signalling, we recorded this processusing time-lapse confocal microscopy in genetically mosaic zebrafish

1
Laboratory of Neurovascular Signaling, Department of Molecular Biology, ULB Neuroscience Institute, Université libre de Bruxelles (ULB), Gosselies, Belgium. 2Max Planck Institute for Heartand Lung Research, ECCPS Bioinformatics and Deep Sequencing Platform, Bad Nauheim, Germany. 3Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala
University, Uppsala, Sweden. 4Mass Spectrometry Facility (MaSUN), University of Namur, Namur, Belgium. 5Center for Microscopy and Molecular Imaging (CMMI), Université libre de Bruxelles(ULB), Gosselies, Belgium. 6Department of Medicine (Huddinge), Karolinska Institutet, Huddinge, Sweden. ✉e-mail: Benoit.Vanhollebeke@ulb.be

Nature | Vol 628 | 25 April 2024 | 863

Articlen = 1960

40

Arterial
BBB

20

LDA
405 nmlaser

Venous
G2/M

0

–20

20

Venous
G1/S

30 hpf–25

t-SNE1

C

tA
s
IS
Vs

0

TC
subpopulation

40

t-SNE2Magnification

80

c

PHBC

fibinafar2
slc16a1azic2b
lrrc15
cpamd8zic2aptprz1bhkdc1
foxq1agpr124 MO
reck MO
wnt7aa MO
β-Catenin LOF

gpr124 MO
reck MO
wnt7aa MO
β-Catenin LOF

gpr124 MO
reck MO
wnt7aa MO
β-Catenin LOF

TC-identity markers

lef1

nkd1

cxcr4aangpt2azic2a

Wnt scoreesm1

TC score

0–2 P 2
Expression

30 hpfgpr124–/–kdrl–/–

36 hpfWT
fli1a:Gal4FF;UAS:GCaMP

β-Catenin
WT
LOF

wnt7aaMO

t-SNE2t-SNE1e

reck
MO

gpr124MO

WTMin. Max.

pex5lagrk5
si:ch1073−90
mamdc2bslco1d1
abcc2
fam210b
popdc3
cyp51
ptdss2
adam15
eif2ak2
tomm20ap3h2
si:ch73-299
FP102309.1

Wnt-activity markers

lef1
fabp11a
AL732488.2
myo1d
mmp25bfdnfatc3alhfpl6dusp8b
cyp1b1
pdgfra24 hpfHindbrain

WT or gpr124 MO

hspa1b
plat
rhoj
kif11

Trunk

ISVs

b

fli1a:Gal4FF;UAS:Kaede

36 hpf

WT
gpr124 MO

n = 23PHBC

100

n = 24

Hindbrain

CNSWT kdrl:EGFP
invading
gpr124 MOTC
kdrl:ras-mCherry
CtAs

TC genotype in mosaic sprouts

a

PHBC
LDA
31 hpfh

636 hpf

WT
mmp25a/b–/–n = 10 18 20 15
P < 0.0001 P = 0.9574
12

24 hpfkdrl:EGFP

42
5

10 15 20 25 30
Time (min)

g

0

5

10 15 20 25 30
Time (min)05

Hindbrain
mmp25b36 hpf

10 15 20 25 30
Time (min)Trunk
mmp25bMerge
kdrl:EGFP

Merge

24 hpf

TC
TC
PHBC

10

DA

Number of sprouts

0

WT

0

mmp25a/b–/–F/F0

gpr124 MO
gpr124 MO
reck MO
reck MO
wnt7aa MO
wnt7aa MO
β-Catenin LOF β-Catenin LOF

8
6
4
2
0
CtAs
36 hpf

ISVs
24 hpfFig. 1 | Mmp25 as a regulator of brain-specific angiogenesis. a, The TC
genotype in mosaic sprouts during brain vascular invasion (36 hpf, n = 23
sprouts) and trunk ISV formation (24 hpf, n = 43 ISVs) of embryos obtained by
five transplantation experiments of WT kdrl:EGFP donor cells into gpr124
MO-injected kdrl:ras-mCherry hosts. b, In vivo photoconversion design of
pre-angiogenic PHBCs. LDA, lateral dorsal aorta. c, t-Distributed stochastic
neighbour embedding (t-SNE) analysis of PHBC EC clusters. d, t-SNE expression
profiles of Wnt–β-catenin target genes and TC markers. Max., maximum; min.,minimum. e, Time-lapse recordings of calcium oscillations in Tg(fli1a:Gal4FF);(UAS:GCaMP7a) PHBCs (31 to 31.5 hpf). f, Wnt-dependent transcripts in 30 hpf

PHBCs or 36 hpf CtAs (β-catenin LOF, IWR-1 treatment). Grey labels below theheat map indicate conditions in which candidate genes are not statisticallydownregulated. μ, mean expression. g, Fluorescent mmp25b WISH andanti-EGFP staining of Tg(kdrl:EGFP) embryos. DA, dorsal aorta. h, Angiogenic
sprouts (arrowheads) in the hindbrain and trunk region of Tg(kdrl:EGFP)
embryos. n ≥ 10 embryos from 4 independent experiments. Data are
median ± interquartile range. P values were calculated using nonparametric
two-tailed Mann–Whitney U-tests. Scale bars, 400 μm (a (left) and b (left)),
100 μm (a (right), e and h), 50 μm (b (right)) and 20 μm (g).

embryos. As in mice8–10, zebrafish brain vascularization is controlled byneural-derived Wnt7a/b ligands, recognized by the atypical Gpr124–
Reck complex of perineural ECs11–14 (Extended Data Fig. 1a). Mid-blastulatransplantation of wild-type (WT) Tg(kdrl:EGFP) cells into gpr124 morpholino (MO)-injected Tg(kdrl:ras-mCherry) hosts resulted in mosaic
perineural primordial hindbrain channels (PHBCs) from which central artery (CtA) sprouts invariably invaded the brain with a WT cellat the tip cell (TC) position11 (Fig. 1a and Supplementary Video 1). By
contrast, peripheral intersegmental vessels (ISVs) were led at equal

frequencies by WT or gpr124-morphant TCs (Fig. 1a). Similarly, after the
CNS-invasive step, gpr124− TCs guiding secondary intraneural vessels
could readily be detected (Extended Data Fig. 1b and Supplementary
Video 2). Wnt–β-catenin signalling therefore appears to be selectively
required in perineural TCs during the initial event of brain invasion.
Within this narrow spatiotemporal window, Wnt signalling could control either TC identity or TC behaviour.
To address this, we transcriptionally profiled fluorescence-activatedcell sorting (FACS)-isolated 30 hours post-fertilization (30 hpf)

864 | Nature | Vol 628 | 25 April 2024

PHBC WT or gpr124 MO ECs after in vivo photoconversion ofTg( fli1a:Gal4FF);(UAS:Kaede) transgenic embryos (Fig. 1b). Single-cell
RNA-sequencing (scRNA-seq) analysis of 144 ECs from WT PHBCs
revealed three major cell clusters (Fig. 1c,d and Extended Data Fig. 1c–e),namely two venous clusters (G2/M and G1/S cell cycle phases, definedby dab2, nr2f2, flt4 and ephb4a) and one arterial cluster (sox7, dll4,
nrp1b, hey2 and efnb2a) containing a TC population (cxcr4a, angpt2a
and esm1). The arterial cluster exhibited a BBB signature (Extended
Data Fig. 1f–h).
The pre-angiogenic PHBCs contained Wnt-positive (lef1, nkd1 andzic2a) and Wnt-negative cells that both contributed to the venous andarterial cell clusters (Fig. 1d). This heterogeneity in Wnt signalling activity was confirmed by the analysis of zebrafish and mouse transgenic
reporters (Extended Data Fig. 1i,j and Supplementary Video 3). TC
and Wnt marker gene expression did not correlate across the PHBC EC
population (Extended Data Fig. 2a). Wnt-independent specification ofperineural TCs was confirmed by implementing a similar scRNA-seq
approach in gpr124 morphants. While, as anticipated, Wnt–β-cateninmarker gene expression was strongly reduced in gpr124 morphants,
TC markers were maintained (Extended Data Fig. 2b).
Wnt–β-catenin signalling has been reported to upregulate Vegfr2
transcripts in the postnatal retinal and brain vasculature15. At brain
invasion stages, the expression levels of kdrl, the main functional
homologue of mammalian VEGFR2 in zebrafish, correlated partiallywith Wnt signalling activity (Extended Data Fig. 2c,d). However, kdrl
expression did not rely on Wnt activity, as it was maintained in gpr124
morphants (Extended Data Fig. 2b,e). Moreover, the formation of the
PHBC-derived basilar artery, a kdrl-dependent process, was unaffectedin gpr124 mutants (Extended Data Fig. 2f), implying that Wnt-deficient
PHBCs remain responsive to VEGF. Furthermore, transgenic endothelial
overexpression of kdrl did not rescue gpr124−/− phenotypes, whereas
it partially corrected the control kdrl−/− phenotypes (Extended Data
Fig. 2g). Finally, in contrast to kdrl−/− vessels, VEGF-induced cytosolic
calcium oscillations16 were unaffected in gpr124−/− Tg( fli1a:Gal4FF);(UAS:GCaMP7a) PHBCs (Fig. 1e, Extended Data Fig. 2h and Supplementary Video 4). Together, these data suggest that the Wnt-dependent
brain-specific angiogenic defects are explained neither by defective
TC specification nor by impaired VEGF signalling.

TC angiodiversity
To identify the angiogenic effectors of Wnt signalling, we performed
bulk RNA-seq analysis of 30 hpf laser-photoconverted FACS-isolated
PHBC ECs in WT as well as gpr124-, reck- and wnt7aa-morphant embryos,
three Wnt–β-catenin loss of function (LOF) conditions (Extended Data
Fig. 3a) that result in avascular brains11,12,17. We also analysed the effectof Wnt inhibition at later stages (36 hpf) by exposing embryos to IWR-1(β-catenin LOF) from 26 hpf onwards (Extended Data Fig. 3b). This strategy identified 40 genes of which the expression was significantly downregulated in at least three Wnt LOF conditions (Fig. 1f and Extended
Data Fig. 3c). Among these candidate genes, known Wnt-target genes
were found (lef1, fabp11a, slc16a1a, zic2a and zic2b), validating theapproach. Five genes were downregulated in all four conditions, including mmp25b, one of the two zebrafish mmp25 paralogues (Fig. 1f andExtended Data Fig. 3d). Low-expressed mmp25a was also downregulated in two Wnt-LOF conditions (Extended Data Fig. 3d). Both paralogues were enriched in the arterial TC cluster (Extended Data Fig. 3e)and correlated with Wnt activity markers (Extended Data Fig. 3f).
Besides in trigeminal, craniofacial and posterior lateral line ganglia18,
mmp25b transcripts were detected by chromogenic whole-mount in situ
hybridization (WISH) in scattered cells of the 36 hpf hindbrain anatomically compatible with PHBCs and CtA TCs (Extended Data Fig. 3g). The
signal in the presumptive ECs was gpr124 dependent, whereas the signal
in sensory ganglia was not (Extended Data Fig. 3g). Combined fluorescence WISH and immunofluorescence staining confirmed the EC- andgpr124-dependent nature of the signal (Extended Data Fig. 3h). Notably,
mmp25b transcripts were detected in hindbrain TCs but were absent in
ISV TCs (Fig. 1g). In the hindbrain, mmp25b expression was higher in TCs
compared with in stalk cells (SCs) and PHBC phalanx cells (Extended
Data Fig. 3i). Consistent with a potential role in brain vascular invasion,
mmp25b expression peaked in 36 hpf CtAs, decreasing thereafter in
PHBC and CtAs (Extended Data Fig. 3j).
Also known as leukolysin19–23, the glycosylphosphatidylinositol(GPI)-anchored Mmp25 has not been linked to angiogenesis or Wnt
signalling to date. To assess Mmp25 function, we generated mmp25a
and mmp25b frameshift alleles in zebrafish using CRISPR–Cas9(Extended Data Fig. 4a,b). Brain angiogenesis was strongly, although
not completely, impaired in double homozygous mmp25a−/−mmp25b−/−(mmp25a/b−/−) mutants (Fig. 1h, Extended Data Fig. 4c and Supplementary Video 5). By contrast, peripheral angiogenesis remained
unaffected (Fig. 1h, Extended Data Fig. 4c–i and Supplementary
Video 6). No other morphological phenotypes were detected in
mmp25a/b mutants. Gene-dosage experiments revealed that mmp25
paralogues exhibit partially redundant functions, with mmp25b−/−
phenotypes aggravated by the gradual loss of WT mmp25a alleles(Extended Data Fig. 5a–c). By contrast, mmp25a−/− and mmp25a−/−b+/−embryos exhibited no vascular defects, the latter of which were used ascontrols in subsequent experiments. Genetic inactivation of mmp25a/b
did not affect VEGF-induced calcium oscillations or Wnt–β-catenin signalling in PHBCs (Extended Data Fig. 5d,e and Supplementary Video 7).
In genetically mosaic embryos, mmp25a/b-mutant cells were outcompeted by WT cells for the TC position selectively in brain-invading
sprouts, but not in trunk ISVs or during secondary sprouting events
within the brain (Extended Data Fig. 5f,g and Supplementary Video 8).
This TC autonomous requirement is reminiscent of the overall function of Wnt signalling in CNS angiogenesis, and compatible withthe membrane retention of Mmp25 through a GPI anchor. Mining
published datasets revealed an evolutionarily conserved expression
pattern in mice, with Mmp25 qualifying as an endothelial-enriched,brain-specific and Wnt-dependent transcript (Extended Data Fig. 6a–j).
MMP25 also contributed to brain vascularization in mice, as constitutive knockouts23 exhibited a partial reduction in angiogenic sprouts inthe embryonic day 10.5 (E10.5) midbrain and forebrain (Extended Data
Fig. 6k). As in zebrafish, the mouse Mmp25 vascular phenotypes were
CNS-specific, with no defects detected in peripheral organs (Extended
Data Fig. 6l–p).

The distinctive MMP repertoire of brain TCs
Vertebrate genomes encode numerous MMPs, some of which are
expressed by ECs. Mmp14 (also known as MT1-MMP) in particular is a well-known TC-enriched angiogenic effector24–27 that, after
TIMP2-dependent activation of Mmp2, amplifies the proteolytic
activity of migrating cells24,25,28–30. While peripheral TCs exhibited theanticipated MMP repertoire (mmp14b+mmp2+), brain TCs lacked mmp2
transcripts, expressing mmp25b instead (Fig. 2a–d). In comparison tommp25b, the expression of mmp14b appeared uniform across PHBC
ECs (Fig. 2a) and was Wnt–β-catenin independent (Fig. 2b). mmp9 was
expressed at low levels in ECs.
Reflecting these distinct expression profiles, brain angiogenesis was largely unaffected by mmp2 or mmp14b genetic inactivation, while robustly reduced in mmp25b or mmp25a/b crispants(Extended Data Fig. 7a,b). However, targeting mmp14b led to a slightbut non-significant reduction in CtAs, which was unaffected by theadditional mutagenesis of mmp2. The combined deletion of mmp25b
and mmp14b modestly aggravated the mmp25b phenotypes, suggesting that mmp14b marginally contributes to brain angiogenesis in an
mmp2-independent manner. In peripheral ISVs, mmp2 and mmp14b
contributed to angiogenic sprouting, while mmp25a and mmp25b
were dispensable (Extended Data Fig. 7c,d). The combined deletion
Nature | Vol 628 | 25 April 2024 | 865

Articlea

mmp25ammp2

mmp14ab

furinaWT (n = 32)

Min. Max.

gpr124 MO (n = 22)

P = 0.0065

P = 0.0391

P = 0.7181

P = 0.3091

P = 0.5687

P = 0.4066

mmp25ammp25bmmp2

mmp9

mmp14ammp14b8

mmp9

mmp25bmmp14bnUMI

6furinb4

t-SNE220
t-SNE1c

mmp25bmmp14b

mmp2

d

mmp9

mmp14bmmp2

mmp9

ISH probe

24 hpf

ISH probe

36 hpfmmp25bMerge

kdrl:EGFP

Merge

kdrl:EGFP

Hindbrain
OV

OV

pBM

12

6
4
2
0
–

mmp25a/b–/–

la

m
a
la 1
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a
la 2
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W
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D
Zn

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8

on

2

10

C

4

Number of CtAs (48 hpf)

gpr124–/–

6

5a
p2
m
m

TC

PHBC

0

* **

n = 31 39 23 25 20 27 20 22+laminin MO
P = 0.0008
P = 0.0009
P > 0.9999
P > 0.9999
P > 0.9999
P < 0.0001
14Merge + DAPIm

ΔZn2+-BD

kdrl:EGFP

36 hpf2+

Catalytic domain
WTg
Laminin-111

8

ΔZ

Cell
membrane

Number of CtA sprouts (38 hpf)

10

Hemopexin
domainGPI anchor

Zn2+-BD(H237A,
H241A, H247A)

P > 0.9999
P > 0.9999
P = 0.0030

f

WT

n = 15 14 18 22 19+mmp25bmRNA

Prodomain
Furincleavage site
(RX[K/R]R)
Catalytic domain
Zn2+-binding
domainHinge

mmp25a/b–/–eFig. 2 | TCs require a specialized MMP repertoire to migrate across the pial
basement membrane. a, t-SNE expression profiles of mmp and furin genes.
b, Expression levels of mmp genes in WT and gpr124-MO PHBC ECs. Dots
represent individual cells. nUMI, normalized unique molecular identifier.
P values were calculated using parametric two-tailed Student’s t-tests.
c,d, Fluorescent mmp25b, mmp14b, mmp2 or mmp9 WISH and anti-EGFP stainingof Tg(kdrl:EGFP) embryos in the hindbrain (c) and trunk region (d). The solidand open arrowheads label mmp-positive and -negative sprouts, respectively.
e, CtA sprouts in 38 hpf Tg(kdrl:EGFP) embryos (n ≥ 14 embryos from ≥3

independent experiments), injected at the one-cell stage with 200 pg ofmmp25b mRNA or its variants (left). BD, binding domain; H-A, H237A, H241A,
H247A. The diagram was created using BioRender. f, Anti-laminin-111
immunofluorescence staining of transverse hindbrain sections, counterstainedwith DAPI. OV, otic vesicle. g, Hindbrain CtAs in Tg(kdrl:EGFP) embryos (n ≥ 20
embryos from ≥3 independent experiments) injected with control or laminin
MOs. In e and g, data are median ± interquartile range. P values were calculatedusing nonparametric Kruskal–Wallis tests. Scale bars, 100 μm (f (left)), 50 μm(c and d) and 10 μm (f (right)).

of mmp2 and mmp14b did not exacerbate the individual phenotypes,
which is compatible with their function in an Mmp2–Mmp14 complex.
In summary, the transcriptional and functional MMP repertoire of TCs
differed between CtAs and ISVs.
To determine whether shifting the brain TC repertoire(mmp25+mmp14+) to a peripheral one (mmp2+mmp14+) is compatible with brain angiogenesis, we injected mRNA encoding mmp25b,
mmp9, mmp2 or variants thereof into mmp25a/b−/− one-cell stageembryos. While the restoration of mmp25b expression partially rescued
mmp25a/b−/− phenotypes, mmp2 or mmp9 did not (Extended Data
Fig. 7e). Mmp2, Mmp9, and Mmp25, produced as zymogens, differ
in their activation mode. While Mmp2 and Mmp9 activation occurs

extracellularly by various proteases, including Mmp1428,29, Mmp25
activation occurs within the secretory pathway, after processing
by furin-like proprotein convertases19–21. Notably, furina is highly
expressed in the PHBC TC cluster, possibly accounting for a robust activation of Mmp25 (Fig. 2a). Moreover, in contrast to the secreted Mmp2
and Mmp9, Mmp25 is retained at the cell surface through a GPI anchor,
which may help to concentrate the proteolytic activity31,32. Assessing
the brain angiogenic potential of mmp2 mRNA variants encoding a constitutively active form of the enzyme (without the prodomain (Pro−)),a GPI-anchored version (GPI+) or both together revealed that only avariant exhibiting the combination of these properties (Pro−GPI+) was
competent for brain angiogenesis (Extended Data Fig. 7e).

866 | Nature | Vol 628 | 25 April 2024

The pial basement membrane obstacle
Brain angiogenesis required an EC-autonomous catalytically active
form of Mmp25, as revealed by mRNA (Fig. 2e) and transgenic endothelial (fli1a promoter) overexpression rescue experiments (Extended Data
Fig. 7f). As Wnt activity, and therefore Mmp25, are selectively required
during the initial step of brain vascular invasion, we reasoned that its
substrate might reside within the pial (or glia limitans) basement membrane (pBM) enwrapping the developing neuroepithelium. The pBM
indeed constitutes a physical barrier that prevents radial overmigrationof neurons and glial cells into the meninges33–37. Reciprocally, it could
therefore represent an obstacle for vascular ingression into the CNS.
Laminin-111 is a well-established structural component of the
pBM34,37–40. Other laminins are found in distinct BMs, including
laminin-411 and laminin-511 around blood vessels39. In 30 hpf zebrafish
embryos and E10.5 mice, anti-laminin-111 antibodies decorated theexternal surface of the developing brain (Fig. 2f and Extended Data
Fig. 7g), thereby defining a landmark interface through which perineural endothelial TCs must navigate, a function that is seemingly
defective in gpr124- and mmp25a/b-mutant zebrafish (Fig. 2f).
MO- and CRISPR–Cas9-based genetic invalidation of lama1, lamb1aor lamc1, the three constitutive chains of laminin-111, partially rescued brain angiogenesis in mmp25a/b−/− embryos, whereas interferingwith lama2, lama4 or lama5 had no effect (Fig. 2g and Extended Data
Figs. 7h–l and 8a,b). These observations suggest that weakening thepBM alleviates the need for Mmp25 in brain angiogenesis. The effect oflaminin-111 inactivation is probably indirect, through a general destabilization of the pBM, as recombinant catalytic domains of zebrafish
and human MMP25 (rzMmp25b and rhMMP25, respectively, Extended
Data Fig. 8c) did not cleave laminin-111 in vitro (Extended Data Fig. 8d),
as previously reported22. Notably, the activity of the recombinant
enzymes was validated on α-1 antitrypsin (Extended Data Fig. 8e), a
known substrate of which the genetic inactivation did not affect mmp25
phenotypes (Extended Data Fig. 8a,b,f,g).

Mmp25-substrate identification
To identify the physiologically relevant Mmp25 substrate within the
poorly characterized pBM, we transcriptionally characterized themeningeal fibroblasts, the main suppliers of pBM proteins41,42. We
first analysed the anatomical relationships between the zebrafishpBM (anti-laminin-111), the meningeal fibroblasts (fluorescencein situ hybridization (FISH) analysis of lama1), a cell population thatis to date uncharacterized in this organism, and the kdrl:EGFP+ ECs(Fig. 3a,b). The laminin-111-positive pBM was evident from 18 hpf
onwards (Fig. 3a and Extended Data Fig. 9a), with the nearest lama1
signal associated with a ventrolateral population of perineural cells(Fig. 3b and Extended Data Fig. 9b). At 18 hpf, these cells probably
represent primary meninx cells, meningeal fibroblast precursors.
Between 24 and 30 hpf, the lama1+ cells flattened out on the ventral
surface of the hindbrain, with the equatorial plane of their oblong
nuclei orienting parallel to the pBM (Fig. 3a,b). Transmission electron
microscopy revealed a typical elongated fibroblast cell morphology(Fig. 3c). These cells resemble pial fibroblasts, although we prefer to
use the broader meningeal fibroblast terminology, as the moleculardiversity of the zebrafish meningeal cell populations remains to beinvestigated.
Using a candidate-based approach, we identified that zebrafish
meningeal fibroblasts expressed col4a5 and col4a6, specific chainsof type IV collagen, in a spatiotemporal pattern very similar to lama1(Fig. 3d,e and Extended Data Fig. 9c–e). Exploration of published mousetranscriptomes confirmed the expression of Col4a5 and Col4a6 byembryonic and adult pial fibroblasts40,42,43 (Extended Data Fig. 9f–j),
together with Col4a1 and Col4a2, common components of EC basement
membranes40,42–44 (Extended Data Fig. 9k). Immunostaining analysis

confirmed the presence of type IV collagen in the laminin-111-positive
E10.5 mouse pBM (Fig. 3f).
We next inactivated the different zebrafish col4 chains by MOs
and somatic CRISPR–Cas9 mutagenesis in mmp25a/b−/− embryos.
Selectively targeting col4a5 or col4a6 rescued the cerebrovascular
defects partially at 36 hpf and nearly completely at 48 hpf (Fig. 3g,hand Extended Data Figs. 8a,b and 10a,b). This finding was confirmed
through Mendelian genetics by crossing the mmp25-mutant alleles
to the dragnet allele45, harbouring a premature stop codon in col4a5(Fig. 3i). The inactivation of col4a5 therefore alleviates the need for
Mmp25 in brain angiogenesis. Notably, brain angiogenesis in dragnet
mutants proceeded as in WT siblings, despite an initial non-significant
reduction in CtA sprouts at 36 hpf (Extended Data Fig. 10c,d).
Recombinant Mmp25 catalytic domains cleaved C-terminally
HA-tagged zebrafish Col4a5 (zCol4a5–HA) in HEK293T lysates, yielding at least four distinct fragments including a major ~30 kDa band
(Fig. 3j). This suggests that the central triple-helix domain of Col4a5
contains an Mmp25-cleavage site, thereby providing a link between
the proteolytic activity of Mmp25 and its function in EC migration
across the pBM.
To identify the cleavage site, we used a two-step strategy as purified Col4a5–Col4a6 heterotrimer is not commercially available. In
step one, we exposed purified human placental collagen IV, which ismostly composed of COL4A1/A2, to rhMMP25. Untreated samples ran
as multiple bands, probably a consequence of the harsh pepsin-based
placental extraction procedure. rhMMP25 cleaved the main bands(Fig. 3k). The two larger parental bands and their presumptive cleavage
products were analysed using mass spectrometry (MS), which revealed
a semi-specific (K)GLPGPPGPPGPYD peptide in rhMMP25-treated collagen IV, containing a C-terminal non-tryptic aspartate (Fig. 3l). By
contrast, the tryptic (K)GLPGPPGPPGPYDIIK fragment ending with alysine residue was found in untreated collagen IV samples. MMPs often
cleave proteins upstream of two small hydrophobic residues, consistent with the observed (Asp)-Ile-Ile cleavage site in human COL4A1,
and the corresponding residues in Col4a1 and Col4a5 across vertebrates (Extended Data Fig. 10e). The peptide mapped to the last shortnon-collagenous region within the central helical part of the α-chain,
upstream of the C-terminal globular domain, essential for collagen
IV sheet formation (Fig. 3l). In step two, to confirm that Mmp25 canprocess Col4a5 or Col4a6 at this site, we expressed tripartite fusion
constructs in Escherichia coli, consisting of an N-terminal glutathione
S-transferase, a C-terminal GFP and a central linker containing the presumptive cleavage site of Col4 chains in tandem with the recognitionsite of the human rhinovirus-derived PreScission enzyme (used as apositive control). Recombinant hMMP25 processed all Col4-based
fusions, while rhMMP2 did not, revealing qualitative differences incollagen IV cleavage by Mmp2 and Mmp25 (Fig. 3m). Such differenceswere confirmed on full-length zebrafish Col4a5, which was cleaved
by both enzymes, but yielded proteolytic fragments of different sizes(Extended Data Fig. 10f).

Uncoupled angiogenesis and BBB formation
Thus far, we have identified that Wnt–β-catenin signalling, by regulating mmp25 expression, enables the migration of TCs across the
Col4a5/6-positive pBM. As Wnt–β-catenin also controls BBB development, this mechanism ensures brain perfusion by vessels led by
TCs of adequate properties. The coupling between brain angiogenesis
and BBB formation therefore appears to rely on the integrity of thepBM. If true, this model implies that, after impairment of the pBM,
Wnt–β-catenin would at least in part become dispensable for brain
angiogenesis. Consistent with this prediction, brain angiogenesis waspartially restored in gpr124 mutants after col4a5 and/or col4a6 inactivation (Fig. 4a and Extended Data Fig. 10g). Under these conditions,brain vessels remained Wnt–β-catenin negative (Fig. 4b and Extended
Nature | Vol 628 | 25 April 2024 | 867

ArticleaLaminin-111
30 hpfb

Merge
kdrl:EGFP
DAPI

lama1

c

Merge
kdrl:EGFP
DAPI

30 hpf

30 hpfHindbrain

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Merge

col4a5Merge

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kdrl:EGFP
DAPI

30 hpfMagnification

Magnification

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NN

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Collagen IV
Isolectin B4

Magnification

Magnificationfg

mmp25a–/–b+/–

E10.5

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+

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rzMmp25b

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rhMMP25

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+

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n= 7

6
4

14

–

b +/

l4
a
co 1
l4
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a
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co
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6

l

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Triple-helix domain

NC

4
Gly-X-Y
interruptions
mmp25a/b–/–

C–

a1

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C–

a1

C–

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a3

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PreScission
a3 a4 a5

a6

a1

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rhMMP25
a3 a4 a5

a6

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rhMMP2a3 a4 a5

a6

70
55
40
35
25

8

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–

100

55

12

0

–/

70

55

2

m

130

100

40

0
mmp25a/b–/–

8

–

8

18

P = 0.0005
P = 0.0781

T

10

7

LFQG
COL4A
GFP
P/GP
peptide 27 kDa
PreScission MMP25cleavage cleavage
site
site
No enzyme

kDa
70
55
40
35
25

kDa
170

130

i

W

31 29 17 18 14 11 15+col4 sgRNAs
P = 0.0004
P = 0.0034
P > 0.9999
P = 0.6707
P > 0.9999
P > 0.9999

Number of CtAs (48 hpf)

12
Number of CtAs (48 hpf)

55 kDa

GST

pCS2+

l4
a
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l4
a5
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14

5a

m

zCol4a5-HA

con = 33Isolectin B4

pBM

k

kDa
180

m
p2

LAMA1 Collagen IV

kdrl:EGFP

48 hpfh

LAMA1

Collagen IV

mmp25a/b–/–
col4a5 sgRNAs

mmp25a/b–/–

LAMA1
DAPI Collagen IV

Trypsin

70
55
40
35
25

Trypsin

KGLPGPPGPPGPYDIIKGEP7055

KGLPGPPGPPGPYD

40
35
25

rhMMP25

Fig. 3 | Mmp25 cleaves meningeal fibroblast-derived Col4a5/6. a,b, Laminin111 immunostaining (a) and lama1 FISH analysis and EGFP immunostaining (b)of Tg(kdrl:EGFP) transverse zebrafish hindbrain sections, counterstained with
DAPI. The arrowheads indicate meningeal fibroblast nuclei. c, Transmission
electron micrograph showing a meningeal fibroblast next to the pBM(arrowhead). N, nucleus. d,e, FISH analysis as in b of col4a5 (d) and col4a6 (e)with EGFP immunostaining. f, Collagen IV and laminin-111 (anti-LAMA1)
co-immunostaining on a E10.5 mouse forebrain and midbrain section,
counterstained with isolectin B4 and DAPI. g,h, Dorsal views (g) andquantification (h) of Tg(kdrl:EGFP) hindbrain CtAs in embryos (n ≥ 11 embryosfrom 3 independent experiments), injected with the illustrated sgRNAs andzCas9 mRNA. i, Hindbrain CtAs in embryos (n ≥ 7 embryos from 4 independent
experiments) crossed to the dragnet col4a5 allele. j, Anti-HA western blot
analysis of zCol4a5–HA-containing HEK293T cell extracts (or control pCS2+

cells) that were treated or not with rzMmp25b or rhMMP25. k, Coomassie blue
staining of human placental collagen IV exposed or not to rhMMP25. The solid
arrowheads indicate parental fragments (black) and rhMMP25 cleavage
products (red) analysed by MS. The open arrowheads indicate additional
differences. l, Collagen IV and its most C-terminal non-tryptic peptide identified
in rhMMP25-treated samples. NC, non-collagenous. m, Anti-GFP western blot
analysis of recombinant GST–GFP fusion proteins (black arrowheads) andcleavage products (white arrowheads). GST and GFP are linked by a PreScission
recognition site and the presumptive cleavage site of MMP25 in COL4A1–6(a1 to a6). C−, GST alone. The diagram was created using BioRender. For h and i,data are median ± interquartile range. P values were calculated using
nonparametric Kruskal–Wallis tests. Scale bars, 500 nm (c (bottom)), 200 μm(f (left)), 100 μm (a, b, d and e (top) and g), 50 μm (f (right)), 20 μm (a, b, d and e(bottom)) and 2 μm (c (top)).

Data Fig. 10h,i) and, accordingly, did not express BBB markers such as
slc2a1a, slc16a1a or fabp11a (Fig. 4c and Extended Data Fig. 10j,k). The
vessels lumenized properly (Fig. 4d and Extended Data Fig. 10l), conducted gata1+ red blood cells (Fig. 4e), assembled vasculatures notably
similar to WT networks (Fig. 4e), but remained leaky to intracardiallyinjected tracers (Fig. 4f). Tracer accumulation did not result from leakage through the impaired pBM, as it was not observed in the absenceof cerebral vessels (Extended Data Fig. 10m). Together, these observations reveal that, after pBM impairment, the organotypic requirementof Wnt signalling for brain vascularization is lost, resulting in properly

868 | Nature | Vol 628 | 25 April 2024

8

gpr124–/–col4a5 MO

Control MO

48 hpf6
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4

Merge

80

80
CtAs at 72 hpf (%)

n =17816910388
100

40

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Reck, Mmp25

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PerineuralECs

Meningeal
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Collagen IV (a5/6)

co

4

n= 6

kdrl:ras-mCherry
36 hpf
Proportion of Wnt– sprouts (%)

3

Wnt–BBB+gata1:DsRed
kdrl:EGFPW
co a5 Tco l4a MO
l4 6
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0

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72 hpf4–

Normalized signal intensity

gpr124–/–col4a5–/–72 hpf8
6

col4a5 MO

Control MO

40

20

Grey mattern = 8 11 8
P = 0.0391
P < 0.0001
5

WT

Wnt+BBB+
60

20

10

kdrl:ras-mCherry–/–

W
CtAs at 48 hpf (%)

slc2a1a
Merge

Neuropil
8 11 8P = 0.0254
P = 0.0008

n=
WT
Dextran 150 kDa 4 dpf

Lumenized
Non lumenizedgpr124–/–n = 82 18 63 39
100

60

gpr124–/–e

slc2a1a+
slc2a1a–
gpr124–/–48 hpf

f

40

0

co
l4
a
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l4
a5 MO
/6
M
O
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l4
a5

T

gpr124–/–d

gpr124–/–col4a5 MO

kdrl:EGFP

60

20

0

WT

7xTCFXla:GFP+
7xTCFXla:GFP–

80

2

c

n = 69 65 101 93
100

CtAs at 48 hpf (%)

gpr124–/– + col4a5 MO

10WT

7xTCF-Xla:GFP

n = 32 38 19 19 15 17
col4 MOs
P = 0.0019
14
P < 0.0001
P < 0.0001
12
P < 0.0001

kdrl:EGFP

48 hpfgpr124–/–b

gpr124–/–

WT

Number of CtAs (48 hpf)agpr124–/–

Fig. 4 | Col4a5/6 inactivation unlocks the quality control of brain
angiogenesis by Wnt–β-catenin signalling. a, Hindbrain CtAs in 48 hpf
Tg(kdrl:EGFP) embryos (n ≥ 15 embryos from ≥3 independent experiments).
Data are median ± interquartile range. P values were calculated using
nonparametric Kruskal–Wallis tests. b, The proportion of 7xTCF-Xla:GFP+ CtAs
in Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) 48 hpf embryos. c, The proportionof slc2a1a-positive CtAs was analysed using fluorescent slc2a1a WISH andanti-EGFP immunostaining in Tg(kdrl:EGFP) embryos. d, The percentage oflumenized CtAs in 72 hpf larvae. For b–d, n = total number of CtAs from 3 (b andc) or 5 (d) independent experiments. e, Wire diagrams (top) and dorsal views(bottom) of 72 hpf Tg(gata1:DsRed);(kdrl:EGFP) larvae. f, FITC 150 kDa dextran
fluorescence intensity 1 h after intracardial injection in larvae at 4 days
post-fertilization (dpf). n ≥ 8 larvae from ≥4 independent experiments.
g, The proportion of 7xTCF-Xla:GFP– CtA TCs in Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryos. The solid and open arrowheads label 7xTCF-Xla:GFP+
and 7xTCF-Xla:GFP– sprouts, respectively. n = 6 independent experiments with
≥6 embryos each. For f and g, data are mean ± s.d. P values were calculatedusing parametric one-way ANOVA. h, Model for brain-specific angiogenesis.
The diagram was created using BioRender. Scale bars, 100 μm (a, b, f and g) and50 μm (c and e).

patterned, yet BBB-defective, cerebrovasculatures. A last prediction
from the pBM-mediated quality control on TCs is that not only gpr124−/−
cells would gain undue access to the CNS after pBM impairment, butalso the naturally occurring Wnt-negative TCs populating the PHBC
(Fig. 1d and Extended Data Fig. 2a,b). Consistent with this prediction,
Wnt-negative TCs invaded the hindbrain of col4a5 and/or col4a6 morphants (Fig. 4g). The pBM therefore appears to control the genetic
competence of the perineural vessels to differentiate into a neuroprotective BBB, by controlling the pioneering TCs. Either vessels are

Wnt competent, displaying the right cohort of cell surface receptors,
and Mmp25-expressing TCs will guide nascent sprouts into the CNS,
or they are not, and they are excluded.

Discussion
Vascular expansion through angiogenesis is a multicellular migration
process that requires the coordinated behaviours of differentially fatedTCs and SCs. TCs invariably guide the nascent angiogenic sprouts andNature | Vol 628 | 25 April 2024 | 869

Article
therefore display common morphological and molecular adaptations
required for tissue exploration. Beyond this apparent uniformity, TCs,
in their task to pervade each vertebrate organ, must navigate throughextracellular spaces of varying composition likely imposing local constraints on TC function. Here we provide an important example of such
TC mechanistic angiodiversity by identifying Mmp25 as a brain-specific
TC angiogenic effector.
An alluring aspect of this brain-specific TC machinery is that
Wnt–β-catenin signalling, besides conferring organ invasive competence, also instructs endothelial tissue-specific adaptations. This
constitutes an elegant quality-control mechanism ensuring that only
BBB-differentiating TCs can guide vessels into the brain. However,neuroprotection implies a uniform activity in Wnt–β-catenin among all
CNS ECs, not only TCs. How and if a quality-control mechanism operates
at the level of naturally occurring Wnt-negative cells other than TCs
remains to be investigated. Notably, studies in the postnatal mouse retina detected such a potential process, in which Wnt-signalling-deficient
Frizzled4−/− ECs were gradually eliminated from genetically mosaic
vessels46.
At the centre of the brain-specific angiogenic program lies the pial(or glia limitans) BM (Fig. 4h). This work extends the function of thiscritical interface between the brain and the periphery. To date, it has
been implicated in proper cortical layering, by acting as a scaffold forradial glia endfeet and avoiding the inside-out overmigration of neurons and glia into the meninges, a condition known in humans as cobblestone lissencephaly34,36,37,47. We show that the pBM also constitutes
an important gatekeeper of the brain by hindering the uncontrolled
ingression of peripheral cells such as leaky ECs. In doing so, here we
reveal a functional connection between two important brain barriers,that is, the BBB and the meningeal barrier to which the pia and its basement membrane belong. This study also illustrates the increasingly
recognized importance of fibroblasts in brain barrier function, bothwithin and at the surface of the brain42,43,48. Besides its angiogenic role
identified in this study, Mmp25 is better known to be expressed byleukocytes, particularly neutrophils19,23. Whether Mmp25 facilitates
immune cell entry across the glia limitans in pathological conditions
like infections, trauma and haemorrhage seems worth pursuing in lightof the results presented here.
Together, we reveal a mechanism by which endothelial TCs gain
brain-specific invasive competence, thereby supporting the existenceof specialized TC angiogenic mechanisms in distinct organs, includingin the bone, liver, retina and solid tumours, where diverse TC morphologies or gene signatures are increasingly reported49,50. Given the essential
role of TCs in guiding new vessels, such organotypic TC functional heterogeneity holds promise for tissue-specific pharmacological control
of angiogenesis, at a level of specificity that is unachievable using thecurrent anti-angiogenic strategies.

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and competing interests; and statements of data and code availability
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Nature | Vol 628 | 25 April 2024 | 871

Article
Methods
Zebrafish strains and husbandry
Zebrafish (Danio rerio) were maintained at 28 °C under a 14 h–10 hlight–dark cycle and raised under standard conditions in a certified
animal facility (LA1500474) in accordance with European and national
ethical and animal welfare guidelines. All of the animal procedures
were approved by the corresponding ethical committee (Commissiond’Ethique et du Bien Être Animal (CEBEA), Université libre de Bruxelles,
protocol approval numbers: CEBEA-IBMM-2016:65 and CEBEA-07 GOS
IBMM). Zebrafish staging was performed as described previously51.
The following published transgenic and mutant lines have beenused in this study: Tg(kdrl:EGFP)s843 (ref. 52), Tg(kdrl:ras-mCherry)s896(ref. 53), Tg(7xTCF-Xla.Siam:GFP)ia4 (ref. 54), Tg( fli1a:Gal4FF)ubs3(ref. 55), Tg(UAS:Kaede)rk8 (ref. 56), Tg(UAS:GCaMP7a)zf415 (ref. 57),
Tg(gata1:DsRed)sd2 (ref. 58), gpr124s984 (ref. 11), wnt7aaulb2 (ref. 17), reckulb3(ref. 59), kdrlhu5088 (ref. 60) and col4a5s510 (ref. 45). The mmp25aulb26 andmmp25bulb27 alleles were generated in this study using CRISPR–Cas9
mutagenesis. All of the zebrafish experiments were performed onembryos and larvae younger than 5 days post-fertilization, before they
became capable of independent feeding.
Mice
Mice were housed at 20 °C under a 12 h–12 h light–dark cycle understandard conditions and were maintained in a certified animal facility(LA1500474) in accordance with European and national ethical and animal welfare guidelines. The relative ambient humidity level ranged from45 to 65%. All animal procedures were approved by the corresponding ethical committee (Commission d’Ethique et du Bien Être Animal(CEBEA), Université libre de Bruxelles, protocol approval number:
CEBEA-08 GOS IBMM). Mice were maintained on the C57BL/6J background and, for experiments, mice of both sexes were used. BAT-GAL
reporter (B6.Cg-Tg(BAT-LacZ)3Picc/J) mice61 and Mmp25-knockout
mice23 were provided by S. Piccolo and C. López-Otín, respectively.
Vascular networks were quantified as the number of CNS-invading
sprouts in the E10.5 midbrain and forebrain in five consecutive 60 μm
sections, and as the organ surface-normalized vascular density (lengthor surface, depending on the vascular morphologies) in 60 μm sectionsof E10.5 forelimbs and E12.5 intestine, stomach, liver and lung.
CRISPR–Cas9-mediated gene disruption in zebrafish
Germline zebrafish mmp25aulb26 and mmp25bulb27 alleles were gen­erated using CRISPR–Cas9 as described previously62. Target sites
were selected using CRISPOR (v.5.01)63. The following primers wereannealed and cloned into the pT7-gRNA vector (Addgene, 46759):
5′-TAGGGGCAATGCCCTGCGAGTG-3′ and 5′-AAACCACTCGCAGG
GCATTGCC-3′ for mmp25a; 5′-TAGGGGACAGCTACAGAGCAAAGA-3′
and 5′-AAACTCTTTGCTCTGTAGCTGTCC-3′ for mmp25b. sgRNAs weresynthesized by in vitro transcription (HiScribe T7 Quick High Yield RNA
Synthesis Kit; New England Biolabs) from BamHI-linearized pT7-gRNA
vectors. Mmp25a was targeted in exon 4 (catalytic domain) and mmp25b
was targeted in exon 2 (pro-domain). Synthetic capped zCas9 mRNA
was transcribed from the XbaI-linearized pT3TS-nls-zCas9-nls vector(Addgene, 46757) using the mMESSAGE mMACHINE T3 Kit (Ambion).
Co-injection of the sgRNAs (30 pg each) and nls-zCas9-nls mRNA
(150 pg) was performed at the one-cell stage.
For somatic gene disruptions, two sgRNAs targeting the sameexon were synthesized using the following primer pairs: mmp2
sgRNA1: 5′-TAGGGGGAACTTTATGATGGGTG-3′ and 5′-AAACCACC
CATCATAAAGTTCCC-3′; mmp2 sgRNA2: 5′-TAGGGGAACTTTATG
ATGGGTGA-3′ and 5′-AAACTCACCCATCATAAAGTTCC-3′; mmp14b
sgRNA1: 5′-TAGGCCAGTCCATTTGATGGAGA-3′ and 5′-AAACTCTCC
ATCAAATGGACTGG-3′; mmp14b sgRNA2: 5′-TAGGATTCCCTGGGAAGTAAGCAT-3′ and 5′-AAACATGCTTACTTCCCAGGGAAT-3′;
mmp25a sgRNA1: 5′-TAGGGGCAATGCCCTGCGAGTG-3′ and 5′-AA

ACCACTCGCAGGGCATTGCC-3′; mmp25a sgRNA2: 5′-TAGGGTCTGGTGAGGCTTATTTT-3′ and 5′-AAACAAAATAAGCCTCACCAGAC-3′;
mmp25b sgRNA1: 5′-TAGGTAGGACTGGTTGAGCCGGTA-3′ and5′-AAACTACCGGCTCAACCAGTCCTA-3′; mmp25b sgRNA2: 5′-TAGG
AGGAGGCAGATATCCATAC-3′ and 5′-AAACGTATGGATATCTGCCTCCT-3′;
lama1 sgRNA1: 5’-TAGGGAACGGCCGTCAGTTCCACT-3′ and 5′-AAAC
AGTGGAACTGACGGCCGTTC-3′; lama1 sgRNA2: 5′-TAGGCGGACTCTGCCACCACAGGT-3′ and 5′-AAACACCTGTGGTGGCAGAGTCCG-3′;
lama1 sgRNA1-scrambled: 5′-TAGGGAACGGCCGTCAGTTACCTC-3′
and 5′-AAACGAGGTAACTGACGGCCGTTC-3′; lama1 sgRNA2scrambled: 5′-TAGGCGGACTCTGCCACCGATGAC-3′ and 5′-AAACG
TCATCGGTGGCAGAGTCCG-3′; lama2 sgRNA1: 5′-TAGGCGCAGAC
AGGCTCCGGTCAG-3′ and 5′-AAACCTGACCGGAGCCTGTCTGCG-3′;
lama2 sgRNA2: 5′-TAGGTCAGCGGGTCACAGCTCAG-3′ and 5′-AAA
CCTGAGCTGTGACCCGCTGA-3′. lama2 sgRNA1-scrambled: 5′-TA
GGCGCAGACAGGCTCCACGGGT-3′ and 5′-AAACACCCGTGGAG
CCTGTCTGCG-3′; lama2 sgRNA2-scrambled: 5′-TAGGTCAGCGGGTCACATGCAGC-3′ and 5′-AAACGCTGCATGTGACCCGCTGA-3′;
col4a1 sgRNA1: 5′-TAGGATAGGTCCTGGCGGTCCGGG-3′ and 5′-A
AACCCCGGACCGCCAGGACCTAT-3′; col4a1 sgRNA2: 5′-TAGGCA
GGTCCCAAAGGAACTGAT-3′ and 5′-AAACATCAGTTCCTTTGG
GACCTG-3′; col4a2 sgRNA1: 5′-TAGGTGGCAGTCCCGGATCTCCAG-3′
and 5′-AAACCTGGAGATCCGGGACTGCCA-3′; col4a2 sgRNA2: 5′-T
AGGAGGTTTGGATGGAGCTTCAG-3′ and 5′-AAACCTGAAGCTCCA
TCCAAACCT-3′; col4a3 sgRNA1: 5′-TAGGAAGGTTGTGCTGGGG
TTCA-3′ and 5′-AAACTGAACCCCAGCACAACCTT-3′; col4a3 sgRNA2:5′-TAGGAAGGATTCCCAGGATTGTGT-3′ and 5′-AAACACACAATCCTGGGAATCCTT-3′; col4a4 sgRNA1: 5′-TAGGTGGGTCGACAGGGCC
CCCAG-3′ and 5′-AAACCTGGGGGCCCTGTCGACCCA-3′; col4a4
sgRNA2: 5′-TAGGAGAACCTTGGGGCCCCTGG-3′ and 5′-AAACCCAGG
GGCCCCAAGGTTCT-3′; col4a5 sgRNA1: 5′-TAGGCCTGGGAAACCTGGAACACC-3′ and 5′-AAACGGTGTTCCAGGTTTCCCAGG-3′; col4a5
sgRNA2: 5′-TAGGCCGGGTTTAAAGGGTCAGCC-3′ and 5′-AAACGGCTGACCCTTTAAACCCGG-3′; col4a6 sgRNA1: 5′-TAGGCTTGGAC
CAGTGGGCAGCGG-3′ and 5′-AAACCCGCTGCCCACTGGTCCAAG-3′;
col4a6 sgRNA2: 5′-TAGGATGGGGGCCCGGGACCAGTT-3′ and 5′-A
AACAACTGGTCCCGGGCCCCCAT-3′; serpina1 sgRNA1: 5′-TAGGTGCTGCCTTGCTGGTAGCAA-3′ and 5′-AAACTTGCTACCAGCAAGGC
AGCA-3′; serpina1 sgRNA2: 5′-TAGGCTGGTAGCAACGGCCTGGG-3′
and 5′-AAACCCCAGGCCGTTGCTACCAG-3′.
The efficiency of somatic gene disruption was scored byhigh-resolution melt analysis (HRMA) using the Illumina Eco real-Time
PCR system, and further characterized using Illumina amplicon deep
sequencing (Azenta Life Sciences).

Genotyping
Zebrafish gpr124s984, wnt7aaulb2, kdrlhu5088, reckulb3 and col4a5s510
and mouse Mmp25 alleles were genotyped as described previously11,17,23,45,59,60. The mmp25aulb26 and mmp25bulb27 alleles were
genotyped by high-resolution melt analysis (Eco Illumina real-time
PCR system) using the following primers: 5′-TTTCCACCTCC
CTCAGTGTC-3′ and 5′-GTGGAAACGCAGAGGTGTGT-3′ for mmp25a;5′-CGCACAGGACAGCTACAGAG-3′ and 5′-CTGCATTTCTCTAATGGC
TCTCTCG-3′ for mmp25b.
MO, RNA and DNA microinjection in zebrafish
MOs targeting gpr124 (4 ng; splice blocking; ACTGATATTGATTTAACTCACCACA)11, reck (0.4 ng; splice blocking; CAGGTAGCAGCCGTCACTCACTCTC)64, wnt7aa (4 ng; splice blocking; TTCCATTTGACCCTACTTACCCAAT)17, lama1 (0.5 ng; translation blocking;
ATCTCCATCATCGCTCAAACTAAAG), lama2 (1 ng; translation blocking GCCACTAAACTCCGCGTGTCCATGT), lama4 (0.5 ng; translation
blocking; GCCATGATTCCCCCTGCAACAACTT), lama5 (0.25 ng;translation blocking; CTCGTCCTGATGGTCCCCTCGCCAT)65, lamb1a(0.125 ng; translation blocking; TATTTCCAGTTTCTTTCTTCAGCGG),

lamc1 (0.125 ng; translation blocking; TGTGCCTTTTGCTATTGCG
ACCTC)66, col4a1 (1 ng; translation blocking; ACACATGGAAGCCG
CATCTTCACAC)67, col4a2 (2 ng; translation blocking; TTCTCACCCTCCATGCGAGCCTAAA), col4a5 (2 ng; translation blocking; ATGTTCCTCTGTTAAGCTAACTGCA), col4a6 (2 ng; translation blocking; AGG
TAAAGTAGGCTATCCTCCTCGT) were obtained from Gene Tools andwere injected at the zygotic stage at the indicated doses. Injection of astandard control MO (CCTCTTACCTCAGTTACAATTTATA, up to 8 ng)
did not affect the brain vasculature.
Transgenic mosaic endothelial overexpression was achievedby co-injecting at the one-cell stage 25 pg of Tol2 transposasemRNA and 25 pg of the pTol2-fli1a:kdrl-2A-nls-mtagBFP2, pTol2fli1a:mmp25b-2A-tagRFP, pTol2-fli1a:mmp25bΔZn2+-BD-2A-tagRFP or pTo
l2-fli1a:mmp25bZn2+-BDH237A,H241A,H247A-2A-tagRFP constructs68.
Capped mRNAs were transcribed in vitro from NotI-linearized
pCS2+ constructs, using the mMessage mMachine SP6 Kit (Thermo
Fisher Scientific) and injected at the one-cell stage at a dose of 200 pg.
The fragment encoding the Zn2+-binding domain (Zn2+-BD; His237–
His247) was deleted in the ΔZn2+-BD mmp25b variant. Three histidines,
essential for Zn2+ chelation, were substituted by alanines in the
Zn2+-BDH237A, H241A, H247A variant, abbreviated as Zn2+-BDH-A. In thePro− mmp2 mRNA variant, the sequences encoding the prodomain(Ala30–Val107) were deleted. The sequences corresponding to the
GPI-anchoring site of Mmp25b (Ser658–Gln697) were fused 3′ to the
mmp2 ORF in the GPI+ mmp2 variant.

Transplantations
Host Tg(kdrl:ras-mCherry)s896 and donor Tg(kdrl:EGFP)s843 embryos
were dechorionated with pronase (Millipore, 53702; 1 mg ml−1) during 5 min at 28 °C in 1/3 Ringer solution, supplemented with penicillin(50 U ml−1) and streptomycin (50 µg ml−1). The embryos were subsequently incubated on agarose-coated dishes in the same medium. At
the mid-blastula stage, 20 to 50 donor cells were transplanted into
the blastoderm margin of stage-matched host embryos. After transplantation, embryos were incubated until the indicated stages. Afterassessing the contribution of EGFP+ transplanted cells using the Leica
M165 stereomicroscope, mosaic vessels were recorded using time-lapse
confocal microscopy. The contribution of cells of a defined genotype
to the TC position was calculated as the fraction of the total numberof mosaic vessels (CtAs or ISVs). The contribution to TC position in
intraneural secondary branches was scored as the fraction of the stalkcell genotype in the initial brain-invading CtA.
Immunofluorescence and in situ hybridization
Zebrafish and mouse embryos were fixed in 4% paraformaldehyde (PFA)in PBS. For sections, embryos were washed in PBS and equilibrated in
30% sucrose in PBS (w/v) overnight at 4 °C. The embryos were thenmounted in 7.5% gelatin (w/v), 15% sucrose (w/v) in PBS and stored at−80 °C. Zebrafish and mouse embryos were cut into 20 and 60 µm
frozen sections, respectively, using the Leica CM1850 Cryostat (Leica)at −30 °C.
For immunofluorescence staining, the sections were washed threetimes with PBS Triton X-100 (0.4%; PBST) for 5 min, blocked using
blocking buffer (PBST, 5% goat serum) for 1 h and then incubated withprimary antibodies in blocking buffer solution overnight at 4 °C. After
three washing steps in PBST for 5 min, the sections were exposed tosecondary antibodies diluted in blocking buffer containing 0.001%
DAPI overnight at 4 °C. After three washing steps in PBST for 5 min,the sections were mounted in DAKO fluorescence mounting medium(Agilent, S3023). The following primary antibodies and lectin wereused: rabbit anti-laminin-111 (Merck, L9393, 1:250, used for zebrafish
immunostaining, polyclonal immunization with an Engelbreth–Holm–
Swarm mouse sarcoma extract), rat anti-laminin-111 (R&D systems,
MAB4656, 1:250, used for mouse immunostainings, monoclonal reactivity towards LAMA1/B1), chicken anti-GFP (Aves Labs, GFP-1020, 1:200),

rabbit anti-collagen type IV (Sigma-Aldrich, AB756P, 1:300), chicken
anti-β-galactosidase (Abcam, ab9361, 1:300), anti-Erg1-Alexa Fluor(AF) 647 conjugate (Abcam, ab196149, 1:250) and isolectin B4-AF594
conjugate (Thermo Fischer Scientific, I21413, 1:200). The following
secondary antibodies were used: goat anti-chicken AF488 (Thermo Fischer Scientific, A11039, 1:500), goat anti-rabbit AF594 (Thermo Fischer
Scientific, A11012, 1:500), and donkey anti-rat AF647 (Thermo Fischer
Scientific, A48272, 1:500).
For in situ hybridization, digoxigenin (DIG)-labelled antisense
riboprobes were produced by in vitro transcription using the
DIG RNA labelling kit and SP6 RNA polymerase (Roche). The templates were amplified from 48 hpf WT embryo cDNA, and cloned
into NcoI/SacI-digested pGEMT using the following primers: kdrl:
5′-GCATGCTCCCGGCCGCCATGGTGGCAGGATTCACTTTGAGTGG-3′
and 5′-CATCCAACGCGTTGGGAGCTCTAGTGTAGGGCTCAATCCGCAG-3′; mmp25b: 5′-ATGAGTTTCTCAGGATATCTTGGTCTGG-3′ and 5′-T
TATTGCGAGTTGAAGCCAATATGAAGC-3′; mmp14b: 5′-GCATGC
TCCCGGCCGCCATGGTGGATGCAGCTCTTCTCTACACG-3′ and 5′-CA
TCCAACGCGTTGGGAGCTCCATGAGGCTGCTGGAAATGTGC-3′; mmp2:5′-GCATGCTCCCGGCCGCCATGGTGCTCACACAGACAAAGAAGTGG-3′
and 5′-CATCCAACGCGTTGGGAGCTCTTTCCTGACATCAGCCGTCC-3′;
mmp9: 5′-GCATGCTCCCGGCCGCCATGGCAAATCTGTGTTCGTG
ACGTTTCC-3′ and 5′-CATCCAACGCGTTGGGAGCTCCTCCTTGATTTGGCAGGCATCG-3′; lama1: 5′-GCATGCTCCCGGCCGCCATGGGTCACAACAAAGCCGACGACTG-3′ and 5′-CATCCAACGCGTTGGGAG
CTCTGAGCGTTCCCTCAGCGCTGT-3′; col4a1: 5′-GCATGCTCC
CGGCCGCCATGGGGTTCTAAGGGTGAAGGAGGTG-3′ and 5′-C
ATCCAACGCGTTGGGAGCTCCCCTCTTCATGCACACTTGAC-3′; col4a2:5′-GCATGCTCCCGGCCGCCATGGCCTAAAGGAGATACCGGACCC-3′ and5′-CATCCAACGCGTTGGGAGCTCCTACAGGTTCTTCATGCACAC-3′;
col4a3: 5′-GCATGCTCCCGGCCGCCATGGGGACAAAAAGGACAGTGTGGTC-3′ and 5′-CATCCAACGCGTTGGGAGCTCGCAAGGTC
ACCTTGAGGCTGTTG-3′, col4a4: 5′-GCATGCTCCCGGCCGCCATGGCTGGGTCCCAGTGGTGCAAAAG-3′ and 5′-CATCCAACGCGTTG
GGAGCTCCATTGGTTGGGGTCATTCATC-3′; col4a5: 5′-GCATGC
TCCCGGCCGCCATGGGGTTTTCCAGGATCTAAAGGAG-3′ and5′-CATCCAACGCGTTGGGAGCTCCGTCCTCTTCATACACACCAC-3′;
col4a6: 5′-GCATGCTCCCGGCCGCCATGGCGTCCAGGAATAATAGGACC-3′ and 5′-CATCCAACGCGTTGGGAGCTCCTACAAGATC
TTCATGCAGAC-3′; slc2a1a: 5′-GCATGCTCCCGGCCGCCATGGCAACTTGGCATTGTCATTG-3′ and 5′-CATCCAACGCGTTGGGAGCTCGG
CTGTGATCTCTTCAAACG-3′; slc16a1a: 5′-GCATGCTCCCGGCCGC
CATGGATGCCTCCAGCAACAGGAGG-3′ and 5′-CATCCAACGCGTTGG
GAGCTCCTATACGACTCCATCTGCCTCCTTTT-3′; fabp11a: 5′-GC
ATGCTCCCGGCCGCCATGGGATCAAATCTCAATTTACAGCTGTTG-3′ and5′-CATCCAACGCGTTGGGAGCTCTTCAAAGCACCATAAAGACTGATAAT-3′. Whole-mount chromogenic in situ hybridizations were performed
as previously described69 using anti-DIG-AP antibodies (Merck,
11093274910, 1:10,000). Combined immunostainings and FISH were
performed as previously described70, using anti-DIG POD antibodies(Merck, 11207733910, 1:1,000) and the TSA Plus Cy3 detection kit (Akoya
Biosciences, NEL744001KT).

Photoconversion and FACS isolation of zebrafish brain ECs
Photoconversion of Tg( fli1a:Gal4FF)ubs3;(UAS:Kaede)rk8 PHBC or CtA
ECs was performed using the Zeiss LSM710 confocal microscope (Carl
Zeiss, objective lenses: Plan-Apochromat ×20/0.8 M27), as described
previously71. In brief, anaesthetized embryos were mounted laterally
in 1% low-melting-point agarose and the fluorescent Kaede protein
was photoswitched by scanning the selected region of interest (ROI)
using a 405 nm laser (five iterations of 50 s). After isolation from theagarose, the embryos were washed in Ca2+/Mg2+-free Hank’s Balanced
Salt Solution (HBSS, Gibco) and dissociated at 28.5 °C for 30 min in
TrypLE select (Thermo Fischer Scientific, 12563011). Dissociation was
stopped by the addition of FBS and centrifugation. The cell pellet wasArticleresuspended in HBSS containing Ca2+/Mg2+ and 5% FBS, filtered andsubmitted for FACS analysis (BD Biosciences FACSAria III).
For scRNA-seq analyses, single photoswitched (red fluorescent)WT ECs were distributed in individual wells of 384-well plates containing 2.3 µl of Smart-seq2 lysis buffer (0.2% Triton X-100, 2 U µl−1
RNase inhibitor, 2 mM dNTP mix and 1 µM Smart-seq2 primer(5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′). The plates werestored at −80 °C before mRNA-seq using the Smart-Seq2 protocol72
and analysis using the Seurat v4 toolkit in Rstudio (v.1.1.463)73. In brief,
single-cell fastq files were demultiplexed by applying standard parameters of the Illumina pipeline (bcl2fastq v.2.19.0.316) using Nextera
XT index kit v2 adapters. Mapping was performed to the zebrafish
reference genome build GRCz11, with TopHat v.2.1.1 and Bowtie1 or
Bowtie2 option. Adapter sequences were removed using Trim Galore
v.0.4.4 before read mapping and doublets were removed using Samtools v.1.16.1 software. The generated BAM files containing the alignment results were sorted according to the mapping position, and raw
read counts for each gene were calculated using the FeatureCounts
function from the Subread package v.1.4.6-p5. For technical control,
92 ERCC RNAs were included in the lysis buffer and in the mapping.
For bulk RNA-seq analyses, Tg( fli1a:Gal4FF)ubs3;(UAS:Kaede)rk8
embryos were injected, or not, at the one-cell stage with gpr124, reck or
wnt7aa MOs and PHBC ECs were isolated at 30 hpf as described above.
Alternatively, embryos were treated with IWR-1 from 26 hpf onwards
and CtA ECs were photoconverted and sorted at 36 hpf, as describedabove. Photoconverted PHBC ECs of 80 embryos were pooled andsubmitted for RNA extraction and RNA-seq, as previously described71.
Transcriptomes were analysed and compared using DESeq2 (v.1.12)74.

resin (Agar 100 resin; Agar Scientific). Resin blocks containing the processed embryos were trimmed to reach the ROI, which was evaluatedby toluidine staining of thin sections (15 μm). Ultrathin 70 nm sections
were then produced with a Leica EM UC6 ultramicrotome and mounted
onto copper-Formvar-carbon grids (Electron Microscopy Sciences).
Observations were made using the Tecnai 10 transmission electron
microscope (FEI), and images were captured with a Veleta camera andprocessed using SIS iTEM v.5.1 software (Olympus).

Light microscopy image acquisition and processing
All images were acquired using the Leica M165 stereomicroscope, theZeiss LSM710 or the Zeiss LSM900 confocal microscope equippedwith the Leica Application Suite (LAS) v.4.2 or ZEN Blue v.3.1 microscopy software. Image analysis was performed using ImageJ v.1.53c.
Zebrafish embryos were imaged live or after fixation in 4% PFA in PBS
overnight at 4 °C. Mouse embryos were fixed (4% PFA in PBS), andstained after sectioning. Live imaging of dechorionated zebrafish
embryos was performed after embryo immobilization with a low doseof tricaine in low-melting-point agarose (1% in E3 zebrafish medium
supplemented with N-phenylthiourea and tricaine) in a glass-bottom
Petri dish (MatTek Corporation). Confocal time-lapse images wererecorded at a stable temperature of 28.5 °C, using an incubationchamber. Ca2+-oscillations were recorded by time-lapse imaging ofTg( fli1a:Gal4FF);(UAS:GCaMP7a) embryos, taking a z stack every 5 sduring the 30 min before CtA sprouting (31–31.5 hpf). Circular ROIs(<5 µm diameter) were centred on oscillating PHBC ECs. F/F0 was calculated to quantify changes in fluorescence, where F0 is the baseline
fluorescence. Ca2+ spikes were identified as events of F/F0 ≥ 1.5.
For angiography, imaging was performed 1 h after injection of 1 nl oftetramethylrhodamine dextran 2,000,000 Da molecular mass (Thermo
Fisher Scientific, D7139, 25 μg µl−1 in PBS) in the heart of 72 hpf larvae
using a micromanipulator. Tracer leakage assays were performed byinjecting 1 nl of 150,000 Da FITC-labelled dextran (FD150S, 25 μg µl−1 inPBS) intracardially and imaging 1 h after injection. Three-dimensional
reconstructions were performed using the Imaris Filament Tracer software (Bitplane) before manual false-colouring to highlight extra- andintracerebral vessels exhibiting or not BBB properties.

Recombinant protein expression and purification
The human MMP25 and MMP2 catalytic domains were amplified from
HUVEC cDNA and the zebrafish Mmp25b catalytic domain was synthesized after codon optimization. The fragments were cloned into the
NcoI and XhoI restriction sites of pET21d. The catalytic domains span
residues Tyr113 to Gly284 of zebrafish Mmp25b (UniProtKB: E7F1N5),
Tyr108 to Gly280 of human MMP25 (UniProtKB: Q9NPA2) and Tyr110
to Asp452 of human MMP2 (UniProtKB: P08253). BL21 (DE3) E. coli cells
were transformed with pET21d-zMmp25b-6xhis, pET21d-hMMP256xhis or pET21d-hMMP2-6xhis and grown in 100–300 ml LB medium(supplemented with 100 µg ml−1 ampicillin). Protein expression was
induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) when
the culture reached an optical density at 600 nm (OD600) of 0.9. Afterovernight incubation at 37 °C under agitation, cells were collectedby centrifugation (5,000g, 20 min, 4 °C) and frozen at −80 °C. After
resuspension in 50 mM Tris (pH 8), cells were mechanically lysed on ice(Microfluidics, 110SCE, 3 cycles). Inclusion bodies were recovered fromthe lysate by centrifugation (16,000g, 20 min, 4 °C) and solubilizedin 8 M urea, 50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM CaCl2 and 50 µm
ZnCl2. The insoluble fraction was removed by centrifugation (16,000g,
20 min, 4 °C) and the supernatant was incubated overnight with 100 µlof Ni+/nitrilotriacetic acid agarose beads (Qiagen) at 4 °C. The beads
were washed with 20 mM imidazole in TBS 8 M urea and elution was
performed with 500 mM imidazole in TBS 8 M urea. Recombinant protein purity was assessed by SDS–PAGE and Coomassie blue staining,and protein concentrations were measured by the BCA protein assay(Thermo Fischer Scientific, 23223). Catalytic domains were refoldedby dilution (1/20, v/v) in 50 mM Tris, 150 mM NaCl, 5 mM CaCl2, 50 µM
ZnCl2, 0.005% Brij-35 (Thermo Fischer Scientific, 20150) for 1 h at 12 °C.
The insoluble fraction was removed by centrifugation (21,400g, 10 min,4 °C). Uncropped gels are available in Supplementary Fig. 1.

Transmission electron microscopy
WT zebrafish embryos (32 hpf) were fixed overnight in 2.5% glutaraldehyde (Electron Microscopy Sciences), 4% PFA at 4 °C and post-fixedwith 1% osmium tetroxide (Electron Microscopy Sciences) and 1.5% ferrocyanide (Electron Microscopy Sciences) in 0.15 M cacodylate buffer.
The embryos were further stained with 1% uranyl acetate (Electron
Microscopy Sciences), serially dehydrated and embedded in epoxy

Western blot analysis
Samples were denatured in Bolt LDS sample buffer and reducing agent(Thermo Fischer Scientific, B0007 and B0009) at 70 °C for 10 min.
Gel electrophoresis was performed using 4–15% Mini-PROTEAN TGX
Precast Protein Gels (Bio-Rad, 4561085). Proteins were transferred tonitrocellulose membranes. After blocking in 5% milk in Tris-bufferedsaline (TBS), the membranes were incubated with primary antibodies(1% milk in 0.05% Tween-20 TBS, TBST) overnight at 4 °C. After washing
in TBST, membranes were incubated with secondary antibodies in 1%
BSA in TBST, for 1 h at room temperature. Blots were revealed using
Western Lightning Plus ECL (PerkinElmer, NEL103001EA).
The following primary antibodies were used: rabbit anti-HA (Merck,
H6908, 1:1,000), chicken anti-GFP (Aves Biolabs, GFP-1020, 1:10,000),
rat anti-laminin-111 (R&D systems, MAB4656, 1:250, monoclonal reactivity towards LAMA1/B1). The following secondary antibodies were used:goat anti-rabbit IgG HRP conjugate (Promega, W401B, 1:5,000), goatanti-chicken IgY HRP conjugate (Thermo Fischer Scientific, A16054,
1:40,000) and rabbit anti-rat IgG HRP conjugate (Merck, A9542,
1:5,000). Uncropped blots are provided in Supplementary Fig. 1.

Mmp25 cleavage assays
For α-1 antitrypsin, 2 µM of α-1 antitrypsin (Athens Research and Technology, 16-16-0011609) was incubated with 2 µM rzMmp25b or 75 nM
rhMMP25 overnight at 28 °C and 37 °C, respectively, in 50 µl Mmp25

cleavage buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 5 mM CaCl2, 0.005%
Brij-35 (Thermo Fischer Scientific, 20150)).
For laminin-111, 15 µg of Matrigel (Corning, 354230) was incubatedovernight at 37 °C with 1 µM of rhMMP25 in 50 µl Mmp25 cleavage
buffer. The samples were concentrated by acetone precipitation
before SDS–PAGE and western blot analysis for LAMA/B1 (R&D systems, MAB4656).
For collagen IV, 20 µg of collagen IV purified from human placenta(Merck, C7521) was incubated with 1 µM rhMMP25 overnight at
37 °C in 50 µl Mmp25 cleavage buffer. The samples were concentra­
ted by acetone precipitation before SDS–PAGE and Coomassie blue
staining.
For recombinant HA-tagged Col4a5 expressed in HEK293T cells,
Zebrafish col4a5 was amplified from 48 hpf zebrafish cDNA, cloned
in fusion to a C-terminal HA tag into pCS2+ (digested with BamHI
and XhoI) and transiently expressed using PEI (polyethylenimine) in
HEK293T cells (ATCC CRL-3216, authenticated by ATCC STR profiling,
tested negative for mycoplasma contamination). The empty pCS2+
was used as negative control. Then, 48 h after transduction, the cells
were washed twice in PBS, before collection and cell disruption using
a disposable grinding pestle in Mmp25 cleavage buffer. After centrifugation (21,400g, 10 min, 4 °C), 4 µg of the supernatant was incubatedovernight with 2 µM of rzMmp25b at 28 °C or 75 nM of rhMMP25 at
37 °C in 50 µl Mmp25 cleavage buffer.
For human COL4A1–6 putative cleavage sites expressed as GST–GFP linkers in E.coli, DNA sequences encoding a N-terminal fusion
between a 12 amino acid fragment centred on the putative cleavage
site of MMP25 in COL4A1–6 and GFP were cloned into pGEX-6P-1
downstream of the GST and the recognition sequence for site-specificcleavage by the PreScission Protease-encoding sequences. BL21
(DE3) E. coli were transformed with these constructs and protein
expression was induced with 1 mM IPTG when OD600 reached 0.7.
After overnight incubation at 30 °C under agitation, cells were collected by centrifugation (5,000g, 20 min at 4 °C) and lysed in 50 mM
Tris (pH 8) using the FastPrep-24 cell disrupter and Lysing Matrix B
Bulk (M.P. Biomedicals). After three cell disruption cycles of 20 s, thecell lysates were clarified by centrifugation (21,400g, 10 min, 4 °C).
Protein concentration of the supernatant was determined using BCA(Thermo Fischer Scientific, 23223). A total of 500 ng of the soluble
fraction was incubated overnight in Mmp25-cleavage buffer with
75 nM of rhMMP25 or rhMMP2 at 37 °C, or with 1 × 10−3 IU of the control PreScission Protease (GenScript, N02799-100) at 25 °C in 50 µl
Mmp25 cleavage buffer. Uncropped gels and blots are available in
Supplementary Fig. 1.

MS analysis
For protein digestion, bands of interest were excised from SDS–PAGE
gels, washed twice with distilled water and shrunk in 100% acetonitrile.
In-gel proteolytic digestion was performed by the addition of 4 µl oftrypsin (Promega; in 50 mM NH4HCO3) and overnight incubation at
37 °C.
For MS, protein digests (supernatants) were analysed using
nano-liquid chromatography–electrospray ionization–MS/MS on
the timsTOF Pro (Bruker v.5.3) system. Peptides were separated bynanoUHPLC (nanoElute, Bruker) on a 75 μm inner diameter, 25 cm C18
column with integrated CaptiveSpray insert (Aurora, IonOpticks) at aflow rate of 200 nl min−1, at 50 °C. LC mobile phase A was 0.1% formic
acid (v/v) in H2O, and mobile phase B was 0.1% formic acid (v/v) in acetonitrile. Digests (1 µl) were loaded at a constant pressure of 600 bar,
directly on the column. After injection of the digest (1 µl), the mobile
phases were linearly increased from 2% B to 13% over 18 min, from 13%
B to 19% over 7 min, from 19% B to 22% over 4 min, and from 22% B to
85% in 3 min.
Data acquisition on the timsTOF Pro was performed using Hystar v.5.1
and timsControl v.2.0. The TIMS accumulation time was 100 ms andmobility (1/K0) ranged from 0.6 to 1.6 V s cm−2. Analyses were performed
using parallel accumulation serial fragmentation (PASEF) acquisition
method75. Per total cycle of 1.1 s, one MS spectrum was followed by ten
PASEF MS/MS spectra.
For data processing, tandem mass spectra were extracted, chargestate deconvoluted and deisotoped by Data analysis (Bruker) v.5.3. All
MS/MS samples were analysed using Mascot (Matrix Science; v.2.8.1),
searching the Human Proteome database (https://www.uniprot.org/uni
protkb?query=(proteome:UP000005640), 101,673 entries) assuming
semi-specific trypsin digestion. Three missed cleavages were tolerated.
Mascot was searched with a fragment ion mass tolerance of 0.050 Da
and a parent ion tolerance of 15 ppm. Carbamidomethyl of cysteine wasspecified as a fixed modification in Mascot. Oxidation of methionine,hydroxylation of lysine and proline, deamination of asparagine andglutamine, and acetylation of the N-terminus were specified in Mascot
as variable modifications.
Peptide and protein identifications were performed using Scaffold(v.Scaffold_5.10.0, Proteome Software). Peptide identifications were
accepted by the Scaffold Local FDR algorithm if establishing a probability higher than 96.0% to achieve an FDR lower than 1.0%. Protein
identifications were accepted if the probability was higher than 5.0% toachieve an FDR lower than 1.0% and containing at least two identified
peptides. Protein probabilities were assigned by the Protein Prophet
algorithm76. Proteins that contained similar peptides and could not be
differentiated based on MS/MS analysis alone were grouped to satisfy
the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters.

Statistics and reproducibility
Seurat v.4 was used to analyse the scRNA-seq datasets. Bulk RNA-seq
data were analysed using DESeq2 v.1.12. Statistical analyses were performed using RStudio v.1.1.463 and GraphPad Prism v.9. Pearson correlation analyses and visualizations were performed using ggcorrplot
v.0.1.3. Normally distributed data are represented as mean ± s.d. andwere analysed using one-tailed one-way ANOVA (with post hoc Dunnett’s test) and two-tailed Student’s t-tests for multiple and single
comparisons, respectively. Non-normally distributed data are represented as median ± interquartile range and were analysed using
one-tailed Kruskal–Wallis tests (with post hoc Dunn’s test) for multiple comparisons and two-tailed Mann–Whitney U-tests for single
comparisons. No statistical methods were used to determine thesample size. The sample size was determined by the technical constraints of the experiments, as well as our and other’s previous work on
zebrafish neurovascular development11–14,17,71. One-cell stage embryos
are undistinguishable irrespective of their genotype, and were therefore randomized during injections. The allocation of organisms intoexperimental groups was randomized. Experimental groups of anexperiment were always raised in parallel, under identical conditions.
For zebrafish and mouse Mendelian genetics experiments, genotyping
was always performed after phenotypic assessment. The researcher
is therefore inherently blinded to the experimental conditions. In MO
and somatic gene disruption experiments, investigators were notblinded. The sex of animals was not determined (embryonic or larvalzebrafish) or was not analysed (embryonic mice) at the developmental
stage of interest. The number and nature of observations (n), mean
or median, type of error bar and statistical tests used for analysis
are indicated in the figure legends. Images of immunofluorescence,in situ hybridization, transmission electron microscopy, and protein
gels or blots are representative of experiments that were repeatedindependently at least three times. All attempts at replication weresuccessful.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.

Article
Data availability
The RNA-seq and MS data were deposited at the NCBI Gene Expression Omnibus through GEO Series accession numbers GSE121041,
GSE233488 and GSE233662 and in PRIDE with the dataset identifier
PXD042613 (Proteomics Identification Database), respectively. Databases used in this study include UniProt (E7F1N5, Q9NPA2 and P08253).
The Human Proteome database (https://www.uniprot.org/uniprotk
b?query=(proteome:UP000005640)) was used for MS/MS analysis.
Publicly accessible datasets used in this study are: ref. 40, (Sequence
Read Archive: PRJNA637987 and http://mousebrain.org/); ref. 42(GEO: GSE150219); ref. 44 (GEO: GSE122564); ref. 77 (GEO: GSE79306);
ref. 78 (GEO: GSE111839); ref. 79 (GEO: GSE95401 and GSE95201); ref. 80(GEO: GSE52564); ref. 81 (GEO: GSE66848); ref. 82 (GEO: GSE74052). Allother data are available in the Article and its Supplementary Information. Source data are provided with this paper.
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Acknowledgements We thank C. Lopez-Otin, A. Inbal, S. Piccolo and H. Baier for sharing
mutant mice and zebrafish; and P. Tebabi, T. Voet, D. Brown, M. Adam, N. Dumont and E. Dupont
for assistance. The illustrations in Figs. 2e, 3m and 4h and Extended Data Fig. 7e were createdusing BioRender. G.S. and L.D.G. are FRIA fellows, and P.C. is a postdoctoral researcher of the
FRS.-FNRS. Work in the B.V. laboratory is supported by FNRS (MIS F.4543.15), the Concerted
Research Action, the Fondation ULB, the H2020 ITN “BtRAIN”, the Queen Elisabeth Medical
Foundation, the FRFS-WELBIO (CR-2017S-05R) and the ERC (Ctrl-BBB 865176). The CMMI is
supported by the European Regional Development Fund and the Walloon Region.
Author contributions All of the authors performed research and/or analysed data, discussed
results and edited the manuscript. G.S. performed most of the experiments, designed
experiments, analysed the data and prepared the figures. P.C., M.A., L.D.G., A.V., B.C., R.F.V.G.
and M.M. performed or assisted in some experiments. S.G., L.H., M. Vanlandewijck and C.B.
performed the bulk and single-cell RNA-seq and provided assistance with the bioinformatic
analysis. M.D. and P.R. performed the MS analyses. M. Vermeersch and D.P.-M. performed theelectron microscopy. B.V. designed the study, supervised the project and wrote themanuscript.
Competing interests The authors declare no competing interests.
Additional information
Supplementary information The online version contains supplementary material available athttps://doi.org/10.1038/s41586-024-07283-6.
Correspondence and requests for materials should be addressed to Benoit Vanhollebeke.
Peer review information Nature thanks Holger Gerhardt, Ghislain Opdenakker and the other,anonymous, reviewer(s) for their contribution to the peer review of this work.
Reprints and permissions information is available at http://www.nature.com/reprints.

Extended Data Fig. 1 | See next page for caption.

Article
Extended Data Fig. 1 | Characterization of the pre-angiogenic perineuralendothelium. a, Lateral views and quantifications of angiogenic sprouts in the
36 hpf hindbrain of WT, wnt7aa−/−, gpr124 −/−, and reck−/− Tg(kdrl:EGFP) embryos(n ≥ 20 embryos from 3 independent experiments, arrowheads: CtA sprouts).
Data represent median ± interquartile range. p-values were determined using
the non-parametric Kruskal-Wallis test. b, TC genotype in mosaic angiogenic
sprouts during post-invasive secondary branching (42 hpf) of embryos
obtained by transplanting wild-type (WT) kdrl:EGFP+ donor cells into gpr124
morpholino-injected kdrl:ras-mCherry hosts. The arrowhead points at a TC
within the hindbrain. n = number of analysed mosaic vessels from 10 embryos
recorded in 5 independent transplantation experiments. c, Heat map of scRNA-

Seq transcriptome analysis of 30 hpf PHBC ECs showing the expression levelsof the 25 highest ranked (lowest p-value) transcripts for each cell cluster. Ven:venous. d-h, t-SNE expression profiles of arterial markers (d), venous markers(e), BBB-associated transporters (f), tight junction proteins (g), and plvapb,
a transcytosis marker (h). i, Lateral view of a WT Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryo at 30 hpf, counterstained with DAPI. The solid andopen arrowheads point at some 7xTCF:GFP+ and 7xTCF:GFP− PHBC ECs,
respectively. j, Immunostaining for β-Galactosidase (LacZ) and Erg1 in atransverse spinal cord section of a WT BAT-GAL E9.5 embryo, counterstainedwith DAPI. The solid and open arrowheads indicate some LacZ+ and LacZ−
perineural vascular plexus (PNVP) ECs, respectively.

Extended Data Fig. 2 | See next page for caption.

Article
Extended Data Fig. 2 | Contrasting Wnt–β-catenin and VEGF signalling
functions during CNS angiogenesis. a, Expression correlation matrix of
Wnt–β-catenin target genes and TC markers based on the scRNA-Seq
transcriptome of the 30 hpf PHBC ECs. Pearson correlation coefficients (r) andp-values were calculated in R. b, Density plots of Wnt–β-catenin target genes,
TC markers and Vegf receptors in WT and gpr124 MO PHBC ECs. nUMI: normalized
unique molecular identifier. p-values were determined using the parametric
two-tailed Student’s t-test. c, t-SNE expression profiles of VEGF receptors within
PHBC ECs. d, Expression correlation matrix between Wnt–β-catenin activity
markers and Vegf receptor genes based on the scRNA-Seq transcriptome of the
30 hpf PHBC ECs. Pearson correlation coefficients (r) and p-values were calculated
in R. e, Chromogenic WISH for kdrl in WT, gpr124 +/−, and gpr124 −/− embryos at 30
hpf. f, Dorsal views of WT, gpr124 −/− and kdrl−/− Tg(kdrl:EGFP) embryos at 48 hpf.
Black arrowheads point at the basilar artery (BA). g, Dorsal views of 48 hpf WT,

gpr124 −/−, or kdrl−/− Tg(kdrl:EGFP) embryos, co-injected or not at the one-cellstage with 10 pg of a pTol2-fli1a:kdrl-2A-nls-mTagBFP2 construct and 25 pg Tol2transposase mRNA. The graphs show CtA numbers and proportion of embryoswith a BA. n ≥ 9 embryos from 8 independent experiments. Data represent
median ± interquartile range. p-values were determined using the two-tailed
non-parametric Mann-Whitney test. h, Characterization of Ca2+ oscillations in
WT, gpr124 +/−, gpr124 −/−, kdrl+/−, or kdrl−/− Tg(fli1a:Gal4FF);(UAS:GCaMP7a) PHBC
ECs at pre-angiogenic stages (31 to 31.5 hpf). Frequency, area under peak, area
under curve, and peak size data are represented as mean ± standard deviation.
p-values were determined using the parametric one-way ANOVA test. The
number of Ca2+-oscillating cells is represented as median ± interquartile range
and analysed with the non-parametric Kruskal-Wallis test. n ≥ 11 embryos from
3 independent experiments.

Extended Data Fig. 3 | See next page for caption.

Article
Extended Data Fig. 3 | RNA-Seq analysis reveals Wnt–β-catenin-dependent
expression of mmp25a/b in CNS ECs. a, Dorsal views of 30 hpf Tg(7xTCF-Xla.
Siam:GFP);(kdrl:ras-mCherry) embryos injected with control, gpr124, reck, or
wnt7aa morpholinos and quantification of 7xTCF:GFP+ cells in the PHBCs.
b, Left, dorsal views of untreated, DMSO, and IWR-1 (20 µM)-treated Tg(7xTCFXla.Siam:GFP);(kdrl:ras-mCherry) embryos at 36 hpf. Centre, number of
7xTCF:GFP+ cells in the PHBCs at 36 hpf. Right, number of CtA sprouts at 36 hpf.
In a,b, n ≥ 10 embryos from 3 independent experiments. Data represent median± interquartile range. p-values were determined using the non-parametric
Kruskal-Wallis test. c, Volcano plots illustrating the differential RNA-Seq
expression profiles between WT and Wnt-deficient PHBC ECs. d, Bulk RNA-Seq
normalized expression values of mmp25a and mmp25b in WT or Wnt-deficient
PHBC ECs. n = brain endothelia analysed by RNA-Seq. Data represent
mean ± standard deviation. In c,d, p-values were determined using DESeq2
analysis. e, scRNA-Seq t-SNE expression profile of mmp25a and mmp25b andtheir combined expression score in PHBC-derived EC clusters. f, Expression
correlation matrix between Wnt–β-catenin signalling target genes and mmp25a,

mmp25b, or their combined score based on the scRNA-Seq transcriptome of
the 30 hpf PHBC ECs. Pearson correlation coefficients (r) and p-values were
calculated in R. g, Dorsal and dorso-lateral views of WISH for mmp25b in WT andgpr124 −/− embryos at 36 hpf. The white arrowhead points at the PHBC, blackarrowheads point at CtA TCs. TG: trigeminal nerve nuclei, CF: craniofacial nerve
nuclei, PLL: posterior lateral line nuclei. h, Combined fluorescent in situ
hybridization for mmp25b and anti-EGFP immunostaining of 30 hpf WT andgpr124 −/− Tg(kdrl:EGFP) embryos. The magnified view shows expression in the
PHBC and the underlying CF. i, Combined fluorescent WISH for mmp25b andanti-EGFP immunostaining of Tg(kdrl:EGFP) embryos in 38 hpf hindbrain when
CtAs harbour two cells. The normalized EC signal intensity is shown on theright. n = 10 angiogenic sprouts from 10 embryos examined over ≥ 3 independent
experiments. j, Normalized mmp25b fluorescence in PHBC and CtAs during
embryogenesis. n ≥ 10 embryos examined from ≥ 3 independent experiments.
In i,j, Data represent mean ± SD. p-values were determined using the parametric
two-tailed Student’s t-test.

Extended Data Fig. 4 | Mmp25 mutagenesis and organotypic requirementin zebrafish blood vessels. a, Sequences of WT mmp25a and the mmp25a ulb26
mutant allele. b, Sequences of WT mmp25b and the mmp25b ulb27 mutant allele.
c, Time-course analysis of the number of CtA and ISV angiogenic sprouts on
one side of WT and mmp25a/b −/− embryos (n ≥ 4 embryos from 3 independent
experiments). d-i, Comparison of the developing vasculature in WT and
mmp25a/b mutant Tg(kdrl:EGFP) zebrafish (n ≥ 10 embryos or larvae from 3

independent experiments) during venous intersegmental vessel sprouting(vISV) at 36 hpf (d), caudal vein plexus angiogenesis (CVP) at 28 hpf (e), subintestinal vein (SIV) sprouting at 34 hpf (f) and 3.5 dpf (g), liver vascularization
at 3 dpf (h), and formation of the myelencephalic choroid plexus (mCP) irrigating
vessels at 54 hpf (i). DLV: dorsal longitudinal vein. PCeV: posterior cerebral vein.
Arrowheads point at angiogenic sprouts. In all panels, data represent median ±.two-tailed Mann-Whitney test.

Article

Extended Data Fig. 5 | See next page for caption.

Extended Data Fig. 5 | Phenotypic characterization of zebrafish mmp25a/
mmp25b mutants. a, Number of CtAs in 36, 40, 48, and 72 hpf embryos andlarvae with different combinations of mutant mmp25a and mmp25b alleles.
b, Number of connections to the basilar artery (BA) at 48 and 72 hpf. Data
represent median ± interquartile range. p-values were determined using thenon-parametric Kruskal-Wallis test. c, Percentage of lumenized CtAs at 48 and72 hpf. Data represent mean ± standard deviation. p-values were determinedusing the parametric one-sided ANOVA. For a-c, n = embryos analysed from ≥ 3
independent experiments. d, Lateral confocal images and quantificationof normalized fluorescence (F/F0) of WT, mmp25a−/−/b +/−, and mmp25a/b −/−Tg( fli1a:Gal4FF);(UAS:GCaMP7a) PHBC ECs at pre-angiogenic stages(31 to 31.5 hpf). For each genotype, images and GCaMP intensity profiles are
representative of ≥ 6 embryos recorded from 4 independent experiments.

e, Dorsal views of 30 hpf WT, gpr124−/−, and mmp25a/b−/− Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry) embryos (n ≥ 8 embryos from 3 independent experiments).
The graph shows the number of 7xTCF:GFP+ PHBC ECs at 30 hpf. Data represent
median ± interquartile range. p-values were determined using the nonparametric Kruskal-Wallis test. f,g, Lateral views of angiogenic sprouts in thehindbrain at the initial step of brain vascular invasion (f, 36 hpf) or at later
intraneural branching steps (g, 42 hpf) and in the trunk region (f, 24 hpf) ofgenetically mosaic embryos obtained by blastula-stage transplantation of
wild-type (WT) kdrl:EGFP + donor cells into mmp25a/b −/− kdrl:ras-mCherry
hosts. White arrowheads point at TCs within the hindbrain. For f and g,
n = distinct mosaic vessels from 8 embryos recorded in 3 independenttransplantation experiments.

Article

Extended Data Fig. 6 | See next page for caption.

Extended Data Fig. 6 | Mmp25 expression and organotypic angiogenic
requirement in mice. a-j, Normalized mouse Mmp25 expression levels inCdh5 + ECs and whole organ extracts at E14.5 (a)77, ECs from different organs(Tie2+) at p7 (b)78, Cdh5 + adult ECs from various organs (c)79, single brain Tie2+
ECs at p7 (d)78, different cell types in the p7 cortex (e)80, brain ECs (CD31+; CD45−)
at E15.5, p5, p9, and in adults (f)44, p7 and adult Tie2+ brain ECs (g)78, brain ECs(CD31+) of control (Cdh5CreERT2;mCherryTRAP) or Axin1 iEC-OE (Axin1+/+;
Cdh5CreERT2;mCherryTRAP) mice at E14.5 (h)81, adult brain ECs (CD31+) of
control (Gpr124flox/+;ROSA-CreER), Gpr124 CKO (Gpr124flox/−;ROSA-CreER),
control (Ctnnb1+/+; Cdh5-CreER), and β-catenin activated (Ctnnb1 lox(ex3)/+; Cdh5CreER) mice (i)82, and adult liver and lung ECs (Cdh5 +) of control (Rosa-BcatGOF) and with β-catenin activated (Rosa-Bcat-GOF; Cdh5-CreERT2) mice ( j)79.

Data represent mean ± standard deviation. In e,f,h-j, n = number of animals
analysed by RNA-seq. k, Isolectin B4 staining of blood vessels on sections(dashed lines) through the E10.5 WT and Mmp25−/− mouse fore- and midbrain.
DAPI: 4′,6-diamidino-2-phenylindole. Magenta asterisks indicate CNSpenetrating angiogenic sprouts. Quantifications are provided on the right.
Data represent median ± interquartile range, and p-values were determinedusing the non-parametric two-tailed Mann-Whitney test. l-p, Comparison of
the vascular networks of WT and Mmp25 KO mice in the E10.5 forelimb buds (l),and the E12.5 intestinal wall (m), gastric wall (n), lung buds (o) and liver (p).
Data represent mean ± SD. p-values were determined using the parametric twotailed Student’s t-test. In k-p, n ≥ 7 embryos from 3 independent experiments.

Article

Extended Data Fig. 7 | See next page for caption.

Extended Data Fig. 7 | Genetic interaction between mmp25 and pial
basement membrane components during brain angiogenesis. a-d, Lateralviews and quantification of 36 hpf hindbrain CtAs (a,b) or 24 hpf trunk ISVs(c,d) in WT Tg(kdrl:EGFP) embryos injected at the one-cell stage with 150 pg ofzCas9 mRNA and 60 pg of the illustrated sgRNAs. e, Left, schematics of Mmp25,
Mmp9 and Mmp2, and its variants. Right, quantification of CtA sprouts in 36
hpf mmp25a/b mutant embryos, injected at the one-cell stage with 200 pg of
the indicated mRNA. The diagram was created using BioRender. f, Transgenic
fli1a-driven endothelial expression of an active or inactive form of Mmp25 in
gpr124 −/− Tg(kdrl:EGFP) embryos. Mmp25 and its variant are expressed as P2AtagRFP fusions. Quantifications of 48 hpf CtAs (arrowheads) are shown on theright. g, Immunostaining of laminin-111 on a section through the E10.5 mouseforebrain and midbrain, counterstained with Isolectin B4 and DAPI. h, Dorsal

views of 48 hpf Tg(kdrl:EGFP) mmp25 mutant embryos injected at the one-cellstage with a control or a lama1-targeting morpholino. i, Quantification ofhindbrain CtAs in 48 hpf embryos, injected at the one-cell stage with 150 pg ofzCas9 mRNA and 60 pg of the illustrated sgRNAs. j, Lateral views of 36 hpf
Tg(kdrl:EGFP) embryos injected at the one-cell stage with a control or a lama1targeting morpholino. k,l, Quantification of hindbrain CtA sprouts in 36 hpf
mmp25a/b mutant embryos, injected at the one-cell stage with a control orlaminin morpholinos (k), or with 150 pg of zCas9 mRNA and 60 pg of theillustrated sgRNAs (l). In all panels, data represent median ± interquartile range.
p-values were determined using the non-parametric Kruskal-Wallis test for
multiple comparisons, and the Mann-Whitney test for the single comparison of
mmp25b vs. mmp25b/mmp14 in b. In b,d-f,i,k,l n ≥ 10 embryos from 3 (b,d,i, k, l),
5 (e), or 6 (f) independent experiments.

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Extended Data Fig. 8 | Somatic gene disruption efficiencies in zebrafish andrecombinant Mmp25 catalytic domains production in E.coli. a,b, Targeting
efficiencies (a) and SNPs and INDELs distributions (b) of embryos co-injectedwith 150 pg of zCas9 mRNA and 60 pg of the indicated targeting sgRNAs, asdetected by Illumina amplicon deep sequencing. c, Coomassie blue staining of
400 ng of recombinant zebrafish and human his-tagged Mmp25 catalytic
domains produced in E.coli, and purified on Ni-NTA resins. rzMmp25b:
recombinant catalytic domain of zebrafish Mmp25, rhMMP25: recombinantcatalytic domain of human MMP25. d, Western blot showing laminin-111 chains(black arrowheads) in 15 µg of Matrigel, after exposure or not to rhMMP25.

e, Coomassie blue staining of 750 ng α1-antitrypsin (α1-AT, 2 µM), after
exposure to 300 ng of rzMmp25b (2 µM) or 12 ng of rhMMP25 (75 nM). The
black arrowheads point to α1-AT degradation products. The open arrowhead
points to rzMmp25b. rhMMP25 is below detection limit. f,g, Quantification ofhindbrain CtAs of control (mmp25a−/−;b +/−) and mmp25a/b −/− Tg(kdrl:EGFP)
embryos at 36 hpf (f) and 48 hpf (g), injected or not (NI) at the one-cell stage
with 150 pg of zCas9 mRNA and 60 pg of a serpina1 (sa1)-targeting sgRNA. Data
represent median ± interquartile range. p-values were determined using thenon-parametric two-tailed Mann-Whitney test. For f and g, n ≥ 10 embryos from
2 independent experiments.

Extended Data Fig. 9 | See next page for caption.

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Extended Data Fig. 9 | Collagen IV isoform expression in zebrafish andmouse meningeal fibroblasts. a, Immunostaining for laminin-111 on transverse
hindbrain sections of 18, 24, and 30 hpf WT Tg(kdrl:EGFP) embryos,
counterstained with DAPI. b, Combined fluorescent in situ hybridization forlama1 and immunostaining for EGFP of 18, 24, and 30 hpf WT Tg(kdrl:EGFP)
transverse hindbrain sections, counterstained with DAPI. In a and b, the 30 hpf
images are the same as in Figs. 3a and 3b, to facilitate the direct comparison
with the earlier stages. c, Combined fluorescent in situ hybridization for
col4a1-6 and immunostaining for EGFP of 30 hpf WT Tg(kdrl:EGFP) transverse
hindbrain sections, counterstained with DAPI. The col4a5 and col4a6 images
are the same as in Figs. 3d and 3e, respectively, to facilitate the comparisonswith the other col4a isoforms. d,e, Combined fluorescent in situ hybridization

for col4a5 (d) or col4a6 (e) and immunostaining for EGFP of 18 and 24 hpf WT
Tg(kdrl:EGFP) transverse hindbrain sections, counterstained with DAPI.
f, Cluster annotation of a whole brain scRNA-Seq analysis during embryonicstages, adapted from La Manno et al.40. g, Cell populations co-expressing thepan-fibroblast marker Col1a2 and the meningeal specific fibroblast markers
S100a6 and Lama1. h, Pial fibroblast clusters and their marker genes as
determined by unsupervised clustering. i, Expression profiles of Col4a1-6
isoforms. j, Normalized expression values for Col4a1-6 in mouse meningeal
fibroblasts (Col1a1+) at E14.5 42. k, Normalized counts for Col4a1-6 transcripts
in ECs (CD31+; CD45−) at E15.5 44. In j and k, Data represent mean ± standard
deviation. n = number of individual meningeal fibroblasts (j) or brain endothelia(k) analysed by transcriptomics.

Extended Data Fig. 10 | See next page for caption.

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Extended Data Fig. 10 | Genetic interaction between col4a5/6 and mmp25 or
gpr124 during brain angiogenesis. a, Quantification of hindbrain CtA sprouts
in 36 hpf mmp25a/b mutant embryos injected at the one-cell stage with 150 pg
of zCas9 mRNA and 60 pg of the indicated col4-targeting sgRNAs. b, Lateralviews and quantification of hindbrain CtA sprouts in 36 hpf Tg(kdrl:EGFP)
mmp25 mutant embryos injected at the one-cell stage with a control or col4targeting morpholinos. c,d, Quantification of hindbrain CtA sprouts in WT,
col4a5 +/− or col4a5−/− embryos at 36 (c) and 48 hpf (d). In a-d, n ≥ 6 embryos from
3 independent experiments. e, Amino acid sequence alignment (Jalview
v2.11.3.2) of the Mmp25 cleavage site (red arrowhead) within human (h), mouse(m), and zebrafish (z) Col4a1 and Col4a5. f, Anti-HA western blot of zCol4a5-HAcontaining HEK293T extracts (or control pCS2+) treated or not with rhMMP25
or rhMMP2. g, Dorsal views of WT, gpr124 and col4a5 single or double mutant
Tg(kdrl:EGFP) embryos at 48 hpf. h,i, Dorsal views (h) and quantification (i) of
7xTCF:GFP+ PHBC ECs in 30 hpf Tg(7xTCF-Xla.Siam:GFP);(kdrl:ras-mCherry)

embryos injected with control, col4a5 and/or col4a6 MOs. n ≥ 10 embryos from
5 independent experiments. In a-d,i, data represent median ± interquartile
range. p-values were determined using the non-parametric Kruskal-Wallis
test. j,k, Combined fluorescent WISH for slc16a1a ( j) or fabp11a (k) andimmunostaining for EGFP in Tg(kdrl:EGFP) embryos injected at the one-cellstage with col4a5 and/or col4a6 MOs. On the right, proportion of slc16a1a or
fabp11a-positive 48 hpf CtAs. In j and k, n = number of CtAs from 3 independent
experiments. l, 2000 kDa dextran-rhodamine angiography in 72 hpf WT andgpr124 −/− Tg(kdrl:EGFP) larvae injected with control or col4a5/6 morpholinos.
m, 1 h before imaging, FITC-labelled 150 kDa dextran was injected intracardiallyinto kdrl−/− 4 dpf (kdrl:ras-mCherry) larvae injected or not with a col4a5-targeting
MO. Tracer accumulation was normalized to the intra-luminal fluorescence ofthe lateral dorsal aorta. n = 11 embryos from 3 independent experiments. Data
represent mean ± SD. p-values were determined using the parametric two-tailed
Student’s t-test.

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