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The Novel Protein, Placenta Endothelial Protein-1 (PEP1), Is A Neural Cue
on Vessel Guidance During Zebrafish Development
Lin ,
YJC Laboratory
NTHU-Medical Science
Figure 1. Analysis of the deduced amino acid
sequence implied the function of PEP1 in cell
motility. (A) PEP1 was predicted to have 11
Thrombospondin type-1 repeats (TSR), one
CD36-binding site, and one RGD site. (B)
Comparison of amino acid sequence of human
PEP1 to zebrafish ortholog shows 69% identities
and 80% similarities. Alignment was calculated by
NCBI webtool-bl2seq and the graphic results was
outputted by MultAlin.
Figure 2. Gene expression of
PEP1 coincides with neural
and cardiovascular system
development. Real-Time qPCR
was performed on RNAs
extracted from zebrafish
embryos (wild-type AB strain)
from 2.75 hour post-fertilization
(hpf) to 72 hpf. Data were
presented as RQ mean value
with standard deviation.
 PEP1 gene was firstly identified and cloned from human placenta.
 In silico domain analysis indicated PEP1 may have functions in
regulating cell motility.
 The expression of zebrafish PEP1 ortholog (zPEP1) begins at 16hpf
during zebrafish embryonic development, right around the stage of
vascular development.
 zPEP1 gene was found to express at developing central neural system,
and coincide spatiotemporally with the expressing pattern of neural stem
cell pan marker, Nestin. These findings imply the participation of PEP1 in
the neurovascular development.
 Morpholino knockdown of zPEP1 in zebrafish embryo caused striking
defects in endothelial cells migration during intersegmental vessel
development, suggesting PEP1 may serve as a guiding cue to regulate
proper angiogenic process in vivo.
 HUVEC with overexpressing c-terminal fragment of PEP1 exhibited
decreased migration ability, while cells treated with anti-PEP1 shRNA
vector showed the opposite result in promoting cell migration.
 Endogenous expression of PEP1 co-localizes with Paxillin, which is a
known focal adhesion factor.
Figure 5. Knockdown of zPEP1 prevents ECs migration during ISV
development. (A, C) The overall development of the morphant is
generally unaffected. (B) At 31-32hpf, sluggish ECs migration was
observed in the morphant in contrast to a well-formed ISV network in the
control embryos (D). The enlarged view indicated ECs line up along the
midline in the morphant (E, F) while ECs in the control (G, H) have fused
with each other to form DLAV. (I) Two morpholinos with different target
sites were used to block mRNA splicing in the morphant. The propagation
of the phenotype was quantified and shown as the chart.
All of our results lead to the conclusion that PEP1 is a novel
angioneurins4, which coordinates the neurovascular interaction.
Morpholino Microinjection
Morpholinos were designed and purchased from Gene Tools, LLC
(Philomath, OR, USA). MOs stock solution was diluted in Danieau buffer
and injected into 1–4 cell stage zebrafish embryos.
Image Analysis
Both zebrafish embryos and HUVEC functional assay were examined
on the Nikon ECLIPE T2000-E inverted fluorescence microscope. Images
were captured using the Evolution-VF CCD camera or Nikon D50 digital
camera. Morphants were compared with the controls collected from the
same clutch of eggs.
Quantitative Real-Time PCR
qRT-PCR was performed using ABI PRIME 7500 Sequence Detection
System, and the data were analyzed using ABI 7500 System SDS Software.
Total RNA was reverse-transcribed into cDNA using SUPERSCRIPT
Ⅲ(Invitrogen). All qPCR products were cloned into pGENTeasy vector
and sequenced.
Whole-mount in situ Hybridization
A Dig-labeled antisense mRNA probe for zPEP1 was used to detect in
situ gene expression. Complementary sense mRNA was used as negative
control. After hybridization with the probe, AP-conjugated anti-Dig
antibody was added and proceeded to react with NBT/BCIP substrate.
P2~P4 HUVEC seeded on glass cover slip were stained with antiPEP1 Ab and paxillin/CD31 Ab, images were examined using Zeiss
LSM510 Meta confocal microscopy.
Materials and methods
Yang ,
of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.
2Division of Cardiology, Department of Medicine, Tao-Yuan Armed Forces General Hospital, Taoyuan, Taiwan
Vascular remodeling and endothelial cell guidance are tightly
regulated processes critical for proper vessel development. Here we report
a novel protein, Placenta Endothelial Protein-1 (PEP1), which participates
in the neurovascular interaction1 and vessel guidance. Identified by SAGE
database mining, PEP1 is highly expressed in human placenta vasculatures
and is well-conserved among all vertebrates. In silico analysis predicted
the potential roles of PEP1 may involve in the cell migration and cell-toECM interaction. In order to characterize PEP1 in vivo, we chose zebrafish
as the model system2 to examine the physiological roles of zebrafish PEP1
ortholog (zPEP1) during embryonic development. Real-time quantitative
PCR analysis first indicated zPEP1 to persistently express after 16 hpf,
which coincides with the start of vascular development. To our surprise,
whole mount in situ hybridization showed that zPEP1 expresses
specifically in the central nervous system, suggesting PEP1 may function
as a neural cue. By injecting morpholino antisense oligos3 to knockdown
zPEP1 expression, we observed prevailing sluggish ISV development in
the morphants. To investigate the molecular mechanism of PEP1,
alteration of PEP1 expression in human umbilical vein endothelial cells
(HUVECs) was done either by over-expression or RNAi knockdown
approaches. As expected, we observed significant effects in HUVEC
migration, reaffirming the involvement of PEP1 in regulating endothelial
cell motility. Furthermore, confocal immunocytochemistry analysis shown
a co-localization of PEP1 and paxillin expression, suggesting the
regulatory effect of PEP1 may be achieved via the paxillin-mediated signal
transduction pathway. Taken together, our study revealed the significant
roles of PEP1 in neurovascular interaction and vascular patterning. Future
analysis on this novel protein shall shed light on the complex
communication network between nervous and vascular systems.
Wang ,
1. Eichmann, A., Makinen, T., and Alitalo, K. (2005). Neural guidance
molecules regulate vascular remodeling and vessel navigation. Genes Dev
19, 1013-1021.
spinal cord
2. Lawson, N.D., and Weinstein, B.M. (2002). In vivo imaging of embryonic
vascular development using transgenic zebrafish. Dev Biol 248, 307-318.
cranial ganglia
pectoral fin
Figure 3. zPEP1 gene expresses in developing nervous system. The
expression of zPEP1 is detectable at 16hpf, locates at CNS and extends to
spinal cord throughout the length of the fish at 24-48hpf (A-D). The
expression at telencephalon, retina, optic tectum, cerebellum, midbrainhindbrain boundary, hindbrain, cranial ganglia, pectoral fin, and heart are
indicated (D-E).
3. Nasevicius, A., and Ekker, S.C. (2000). Effective targeted gene
'knockdown' in zebrafish. Nat Genet 26, 216-220.
Figure 4. zPEP1 gene expression co-localizes with neural stem cell
pan marker, Nestin. The expression pattern of zPEP1 (A, C)
corresponds with Nestin (B, D) at cranial ganglia, telecephalon, retina,
MHB, and spinal cord at 24hpf and 36hpf.
Figure 6. PEP1 modulated HUVECs motility in the transwell migration
assay. (A) Cell overexpressing c-terminal fragment of PEP1 exhibited
decreased migration ability, whereas cells treated with empty vector
behaved similar to the mock. (B) Cells infected with anti-PEP1 shRNA
lentivirus exhibited higher migration ability.
Figure 7. HUVEC
showed PEP1 was colocalized with paxillin.
HUVEC were stained with
PEP1 (A), paxillin (B), and
DAPI (C), the merged
images were shown in (D).
PEP1 was co-localized with
paxillin in focal adhesion
4. Zacchigna, S., Lambrechts, D., and Carmeliet, P. (2008). Neurovascular
signalling defects in neurodegeneration. Nat Rev Neurosci 9, 169-181.
We acknowledged financial support from the National Science Council,
Taiwan. (95-2311-B-007-021-MY3 to Y.-J.C.)
Contact information
Chieh-Huei Wang (First Author)
Department of Life Science & Institute of
Bioinformatics and Structural Biology
National Tsing Hua University
Address: No.101, Sec.2, Kuang Fu Road, Hsinchu,
30013, Taiwan, R.O.C.
Office: LS-2, Room 403
Tel: 886-3-571-5131 ext.33451
Fax: 886-3-571-5934
E-mail: [email protected]
Y.-J. Chuang Ph.D. (Corresponding Author)
Assistant Professor
Department of Medical Science & Institute of
Bioinformatics and Structural Biology
National Tsing Hua University
Address: No.101, Sec.2, Kuang Fu Road, Hsinchu, 30013,
Taiwan, R.O.C.
Office: LS-2, Room 410
Tel: 886-3-574-2764
Fax: 886-3-571-5934
E-mail: [email protected]