GIPC proteins negatively modulate Plexind1 signaling during vascular development

  1. Jorge Carretero-Ortega  Is a corresponding author
  2. Zinal Chhangawala
  3. Shane Hunt
  4. Carlos Narvaez
  5. Javier Menéndez-González
  6. Carl M Gay
  7. Tomasz Zygmunt
  8. Xiaochun Li
  9. Jesús Torres-Vázquez  Is a corresponding author
  1. New York University Langone Medical Center, United States
  2. New York University School of Medicine, United States
7 figures, 1 table and 9 additional files

Figures

PLXND1’s C-terminal GBM and GIPC's GH1 and PDZ domains are the key molecular determinants of the PLXND1-GIPC interaction.

(A) Diagrams of the wild-type (WT) and truncated V5-tagged (red) forms of the cytosolic tail of murine PLXND1 (V5-C-mPLXND1) used for co-immunoprecipitation experiments. Color-coding is used to …

https://doi.org/10.7554/eLife.30454.002
Figure 2 with 2 supplements
The plxnd1skt6 allele encodes a functional Plxnd1 receptor putatively impaired in GIPC binding, and its homozygosity induces angiogenesis deficits with low frequency.

(A, B) Diagrams of the cytosolic tails of the zebrafish Plxnd1 proteins encoded by the WT (A) and plxnd1skt6 mutant (B) alleles including their C-terminal amino acid sequences. Color-coding is used …

https://doi.org/10.7554/eLife.30454.003
Figure 2—figure supplement 1
Penetrance and expressivity of Se-DLAV truncations in plxnd1skt6 mutants at 32 hpf.

(A) Penetrance bar graph. Percentage of embryos of the indicated genotypes with Se-DLAV truncations (gray; a summation of the ‘minimal,’ ‘moderate,’ and ‘maximal’ categories) and with non-truncated …

https://doi.org/10.7554/eLife.30454.004
Figure 2—figure supplement 2
A Plxnd1 form deficient in GIPC binding because of deletion of the receptor’s GBM (Plxnd1ΔGBM) is active in vivo.

(A, B) Diagrams of the GAL4-responsive constructs used for forced endothelial expression in plxnd1fov01b; Tg(fli1a:GAL4FF)ubs4; Tg(flt1:nls-mCherry)skt7 embryos. (A) Construct for expression of the …

https://doi.org/10.7554/eLife.30454.005
Figure 3 with 1 supplement
plxnd1skt6 mutants are hypersensitive to the antiangiogenic drug SU5416.

(A–D) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. …

https://doi.org/10.7554/eLife.30454.006
Figure 3—figure supplement 1
Penetrance and expressivity of Se-DLAV truncations in DMSO-treated and SU5416-treated WT and embryos and plxnd1skt6 mutants at 32 hpf.

(A) Penetrance bar graph. Percentage of embryos of the indicated combinations of genotypes and treatments with Se-DLAV truncations (gray; a summation of the ‘minimal,’ ‘moderate,’ and ‘maximal’ …

https://doi.org/10.7554/eLife.30454.007
Figure 4 with 3 supplements
gipc mutants display angiogenesis deficits.

(A) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. …

https://doi.org/10.7554/eLife.30454.008
Figure 4—figure supplement 1
GIPC proteins encoded by both the wild-type and mutant gipc1, gipc2, and gipc3 alleles.

In the mutant proteins, the red bar denotes novel amino acid sequences resulting from mutagenic frameshift(s). See Supplementary file 1 and Supplementary file 8. Related to Figure 4.

https://doi.org/10.7554/eLife.30454.009
Figure 4—figure supplement 2
Angiogenesis deficits of gipc1skt1 and gipc2 skt3/skt4 mutants at 32 hpf.

(A–C) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. …

https://doi.org/10.7554/eLife.30454.010
Figure 4—figure supplement 3
Penetrance and expressivity of Se truncations in gipc mutants at 32 hpf.

(A) Penetrance bar graph. Percentage of embryos of the indicated genotypes with Se truncations (gray; a summation of the ‘severe,’ ‘medium,’ and, ‘weak’ categories) and with non-truncated Se (black; …

https://doi.org/10.7554/eLife.30454.011
Figure 5 with 1 supplement
plxnd1 heterozygosity suppresses the angiogenesis deficits of gipc1skt1(MZ) mutants.

(A, B) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. …

https://doi.org/10.7554/eLife.30454.012
Figure 5—figure supplement 1
Penetrance and expressivity of Se-DLAV truncations in gipc1skt1(MZ) and gipc1skt1(MZ); plxnd1fov01b/+ embryos at 32 hpf.

(A) Penetrance bar graph. Percentage of embryos of the indicated genotypes with Se-DLAV truncations (gray; a summation of the ‘minimal,’ ‘moderate,’ and ‘maximal’ categories) and with non-truncated …

https://doi.org/10.7554/eLife.30454.013
Removal of plxnd1 activity from gipc1skt1(MZ); gipc2skt4(MZ) maternal-zygotic (MZ) double mutants yields a phenotype similar to that of plxnd1 nulls.

(A–D) Confocal lateral images of the trunk vasculature (green) of 32 hpf embryos (region dorsal to the yolk extension). Anterior, left; dorsal, up. Scale bars (white horizontal lines), 100 μm. …

https://doi.org/10.7554/eLife.30454.014
Figure 7 with 4 supplements
GIPC depletion potentiates SEMA3E-induced, PLXND1-dependent ERK inactivation in HUVEC/TERT2 cells.

(A–D) Representative Western blot of active ERK1/2 (pERK) and total ERK1/2 (ERKTotal) from total cell lysates of HUVEC/TERT2 cells under the four conditions (shRNA and gRNA combinations) and the …

https://doi.org/10.7554/eLife.30454.015
Figure 7—figure supplement 1
GIPC depletion potentiates SEMA3E-induced, PLXND1-dependent ERK inactivation in HUVEC/TERT2 cells.

(A, B) Bar graphs. Means of percentual relative ERK activity (pERK/ERKTotal) under the described conditions and SEMA3E treatments. Error bars, ± SEM. Relative ERK activity. (A) Conditions: control …

https://doi.org/10.7554/eLife.30454.016
Figure 7—figure supplement 2
Efficient shRNA-mediated knockdown of GIPCs and CRISPR/Cas9-mediated knockout of PLXND1 in HUVEC/TERT2 cells.

(A–H) Western blots for GIPC1-2, PLXND1, and GAPDH (loading control) from TCLs of stable cells demonstrating the effective decrease of GIPC1-2 and PLXND1 levels. (A–D) TCLs from non-targeting gRNA#1 …

https://doi.org/10.7554/eLife.30454.017
Figure 7—figure supplement 3
GIPC knockdowns potentiate the SEMA3E-induced/PLXND1-dependent cell collapse of primary HUVEC.

(A–F). Representative fluorescent images of HUVEC morphology in cell collapse experiments under the following conditions. No ligand (A–C; top) or 45 min stimulation with 10 nM of SEMA3E (D–F; …

https://doi.org/10.7554/eLife.30454.018
Figure 7—figure supplement 4
Efficient shRNA-mediated GIPC (GIPC1, GIPC2, and GIPC3) and PLXND1 knockdowns in primary HUVEC used for cell collapse experiments.

Western blots for GIPC1, GIPC2, PLXND1, and GAPDH (loading control) from TCLs of cells infected with the indicated shRNA lentiviral particles. Note the effective decrease of GIPC1-2 and PLXND1 …

https://doi.org/10.7554/eLife.30454.019

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(Danio rerio)
Tg(kdrl:HsHRAS-mCherry)s896DOI:10.1101/gad.1629408ZFIN ID: ZDB-
ALT-081212–4
Transgenic insertion
Genetic reagent
(Danio rerio)
Tg(fli1a:EGFP)y1PMID:12167406ZFIN ID: ZDB-
ALT-011017–8
Transgenic insertion
Genetic reagent
(Danio rerio)
Tg(fli1a:GAL4FF)ubs4DOI:10.1016/j.devcel.
2011.06.033
ZFIN ID: ZDB-
ALT-110921–1
Transgenic insertion
Genetic reagent
(Danio rerio)
Tg(flt1:nlsmCherry)skt7This paperTransgenic insertion.
Made using Torres-Vázquez
lab plasmid #1208
Genetic reagent
(Danio rerio)
gipc1skt1This paperPutative null mutant allele
Genetic reagent
(Danio rerio)
gipc1skt2This paperPutative null mutant allele
Genetic reagent
(Danio rerio)
gipc2skt3This paperPutative null mutant allele
Genetic reagent
(Danio rerio)
gipc2skt4This paperPutative null mutant allele
Genetic reagent
(Danio rerio)
gipc3skt5This paperPutative null mutant allele
Genetic reagent
(Danio rerio)
plxnd1fov01bPMID: 11861480
DOI:10.1016/j.devcel.
2004.06.008
ZFIN ID: ZDB-ALT
-010621–6
Null mutant allele
(point mutation)
Genetic reagent
(Danio rerio)
plxnd1skt6This paperHypermorphic mutant
allele
Cell line
(Cercopithecus aethiops)
COS-7 (Monkey
Kidney Fibroblasts)
American Type
Culture Collection
Cat. #CRL-1651.
RRID:CVCL_0224
https://www.atcc.org/products/All/CRL-1651.aspx
Cell line
(Homo sapiens)
HUVEC/TERT2
(Immortalized Human
Umbilical Vein Endothelial
Cells)
American Type
Culture Collection
Cat. #CRL-4053.
RRID:CVCL_9Q53
https://www.atcc.org/Products/All/CRL-4053.aspx
Cell line
(Homo sapiens)
HUVEC (Normal
Primary Human Umbilical
Vein Endothelial Cells)
Lifeline Cell TechnologyCat. #FC-0003https://www.lifelinecelltech.com/shop/cells/human-endothelial-cells/umbilical-vein-endothelial-cells/huvec-fc-0003/
Cell line
(Homo sapiens)
Non-targeting gRNA1.
Pool of HUVEC/TERT2
cells.
This paperDerived from HUVEC/TERT
2 cell line (ATCC CRL4053).
Cells were grown under blasticidin
(4 μg/ml) selection and used
between 7th and 10th passages.
Cells stably coexpress
Cas9 nuclease and non-targeting
gRNA1 (from Torres-Vázquez
lab plasmid #1859)
Cell line
(Homo sapiens)
Non-targeting gRNA2.
Pool of HUVEC/TERT2
cells.
This paperDerived from HUVEC/TERT
2 cell line (ATCC CRL4053).
Cells were grown under
blasticidin (4 μg/ml) selection
and used between 7th-10th
passages.
Cells are stably coexpressing
Cas9 nuclease and non-
targeting gRNA2 (from
Torres-Vázquez lab plasmid #1860)
Cell line
(Homo sapiens)
PLXND1 gRNA KO1.
Monoclonal PLXND1 KO
HUVEC/TERT2 cell line.
This paperBiallelic (transheterozygous)
PLXND1 knockout line. Derived
from HUVEC/TERT 2 cell line
(ATCC CRL4053). Cells were
grown under blasticidin (4 μg/ml)
selection and used between
7th-10th passages.
Cells are stably coexpressing
Cas9 nuclease and PLXND1
gRNA KO1 (from Torres-Vázquez
lab plasmid #1846)
Cell line
(Homo sapiens)
PLXND1 gRNA KO2.
Monoclonal PLXND1 KO
HUVEC/TERT2 cell line.
This paperBiallelic (transheterozygous)
PLXND1 knockout line. Derived
from HUVEC/TERT 2 cell line
(ATCC CRL4053). Cells were
grown under blasticidin (4 μg/ml)
selection and used between
7th-10th passages.
Cells are stably coexpressing
Cas9 nuclease and PLXND1
gRNA KO2 (from Torres-Vázquez
lab plasmid # 1847)
Cell line
(Homo sapiens)
HEK293T
(embryonic kidney cells)
Matthias
Stadtfeld lab, NYU
Recombinant
DNA reagent
V5-C-mPLXND1WTThis paperTorres-Vázquez lab
plasmid #862. Vector
backbone: pcDNA3.1/
nV5-DEST-V5
Recombinant
DNA reagent
V5-C-mPLXND1ΔCYSEAThis paperTorres-Vázquez lab
plasmid #863. Vector
backbone: pcDNA3.1/
nV5-DEST-V5
Recombinant
DNA reagent
V5-C-mPLXND1ΔGBMThis paperTorres-Vázquez lab
plasmid #1774. Vector
backbone: pcDNA3.1/
nV5-DEST-V5
Recombinant
DNA reagent
FLAG-mGIPC1WTDOI:10.1091/mbc.12.3.615Torres-Vázquez lab
plasmid #864. Vector
backbone: pFLAG-CMV1
Recombinant
DNA reagent
FLAG-mGIPC1GH1DOI:10.1091/mbc.12.3.615Torres-Vázquez lab
plasmid #868. Vector
backbone: pFLAG-CMV2
Recombinant
DNA reagent
FLAG-mGIPC1PDZDOI:10.1091/mbc.12.3.615Torres-Vázquez lab
plasmid #866. Vector
backbone: pFLAG-CMV3
Recombinant
DNA reagent
2xHA-Plxnd1WTThis paperGAL4-responsive,
Gateway and IRES-based
bicistronic vector for Tol2-
mediated zebrafish transgenesis.
Torres-Vázquez lab plasmid #1414
Recombinant
DNA reagent
2xHA-Plxnd1ΔGBMThis paperGAL4-responsive, Gateway
and IRES-based bicistronic
vector for Tol2-mediated
zebrafish transgenesis.
Torres-Vázquez lab plasmid #1685
Recombinant
DNA reagent
lentiCRISPR v2-BlastAddgeneCat. #83480A gift from Mohan Babu.
https://www.addgene.org/83480/
Recombinant
DNA reagent
Non-targeting gRNA1This paperTorres-Vázquez lab
plasmid #1859. Vector
backbone: lentiCRISPR
v2-Blast
Recombinant
DNA reagent
Non-targeting gRNA2This paperTorres-Vázquez lab
plasmid #1860. Vector
backbone: lentiCRISPR
v2-Blast
Recombinant
DNA reagent
PLXND1-KO1This paperTorres-Vázquez lab
plasmid #1846. Vector
backbone: lentiCRISPR v2-Blast
Recombinant
DNA reagent
PLXND1-KO2This paperTorres-Vázquez lab
plasmid #1847. Vector
backbone: lentiCRISPR v2-Blast
Recombinant
DNA reagent
Control shRNA Lentiviral
Particles-A (Non-targeting
control shRNA)
Santa Cruz
Biotechnology
Cat. #sc-108080Encodes a non-targeting
shRNA sequence, will not
lead to the specific degradation
of any known cellular mRNA
Recombinant
DNA reagent
GIPC shRNA (h)
Lentiviral Particles
Santa Cruz
Biotechnology
Cat. #sc-35475-VshRNA pool (three
target-specific constructs
against human GIPC1 that
encode 19–25 nt
(plus hairpin) shRNAs).
Target sequences
(sense sequences 5’ to 3’):
(1) CUGACGAGUUCGUCUUUGA
(2) CCACCACUUUCCACCAUCA
(3) CUGAAUUUGCUGUCUUGAA
Recombinant
DNA reagent
GIPC2 shRNA (h)
Lentiviral Particles
Santa Cruz
Biotechnology
Cat. # sc-75132-VshRNA pool (three
target-specific constructs
against human GIPC2 that
encode 19–25 nt (plus hairpin)
shRNAs). Target sequences
(sense sequences 5’ to 3’):
(1) CAGACGAAUUUGUCUUUGA
(2) GGACACCUUUACUAACUCU
(3) CCAACUUUCUCUCUUUGUA
Recombinant
DNA reagent
GIPC3 shRNA (h)
Lentiviral Particles
Santa Cruz
Biotechnology
Cat. #sc-62376-VshRNA pool (three
target-specific constructs
against human GIPC3 that
encode 19–25 nt (plus hairpin)
shRNAs). Target sequences
(sense sequences 5’ to 3’):
(1) CCUUCAUCAAGAGAAUCAA
(2) GGAGUUUGCACGCUGUUUA
(3) GACAAGUUCCUCUCUAGAA
Recombinant
DNA reagent
Plexin-D1 shRNA
(h) Lentiviral Particles
Santa Cruz
Biotechnology
Cat. #sc-45585-VshRNA pool (three
target-specific constructs
against human PLXND1 that
encode 19–25 nt (plus hairpin)
shRNAs). Target sequences (sense
sequences 5’ to 3’):
(1) GUCAAGAUAGGCCAAGUAA
(2) CCAUGAGUCUCAUAGACAA
(3) CCACAGACAGUUUCAAGUA
Sequenced-based
reagent
plxnd13207-3462 morpholinoDOI:10.1016/j.devcel.
2004.06.008
Validated splice-blocking
morpholino against zebrafish
plxnd1. Synthesized by
GENE TOOLS, LLC).
Sequence (5’ to 3’):
CACACACACTCACGTTGATGATGAG
AntibodyChicken anti-GFPInvitrogenCat. #A10262IF (1:1,000); zebrafish
AntibodySheep anti-mCherryHolger Knaut lab, NYUIF (1:1,000); zebrafish.
Custom made antibody
AntibodyMouse anti-pFAK Tyr397MilliporeCat. #05–1140IF (1:1,000); zebrafish
AntibodyRabbit anti-GIPC1Proteintech GroupCat. #14822–1-AP.
RRID:AB_2263269
WB (1:3,000). This antibody
detects GIPC1, GIPC2,
and GIPC3 (our data)
AntibodyRabbit anti-GIPC2AbcamCat. #ab175272WB (1:5,000). This antibody
detects GIPC1 and
GIPC2 (our data)
AntibodyRabbit anti-GIPC3AbcamCat. #ab186426WB (1:5000). This antibody
is specific for GIPC3 (our data).
Validated against HeLa TCL
(positive control; a gift from
Mamta Tahiliani’s lab, NYU)
AntibodyMouse anti-PLXND1R and D SystemsCat. #MAB41601
Clone #752815
WB (1:250). Lyophilized
reagent reconstituted in
200 μl of sterile PBS
(GIBCO, Cat. #10010–023)
AntibodyRabbit anti-Phospho-
p44/42 MAPK (Erk1/2)
(Thr202/Tyr204) (D13.14.4E)
XPTM
Cell Signaling TechnologyCat. #4370S.
RRID:AB_2315112
WB (1:20,000)
AntibodyMouse anti-p44/42
MAPK (Erk1/2) (L34F12)
Cell Signaling TechnologyCat. #4696SWB (1:10,000)
AntibodyRabbit anti-GAPDH
(D16H11)
Cell Signaling TechnologyCat. #5174P.
RRID:AB_10622025
WB (1:20,000)
AntibodyMouse anti-FLAG M2SIGMA-ALDRICHCat. #F3165,
clone M2.
RRID:AB_259529
WB (1:20,000)
AntibodyRabbit anti-V5-Tag
(D3H8Q)
Cell Signaling TechnologyCat. #13202S.
RRID:AB_2687461
WB (1:10,000)
Peptide,
recombinant protein
Human Semaphorin 3ER and D SystemsCat. #3239-S3BWorking concentration
of 2 nM (prepared in 1xPBS
with 0.1%BSA (SIGMA_ALDRICH,
Cat.A8022)
Chemical
compound, drug
SU5416SIGMA-ALDRICHCat. #S8442Working concentration
of 0.2 μM in fish water.
From 10.5 mM stock solution
in DMSO (SIGMA-ALDRICH,
Cat. #D8418)
Chemical
compound, drug
Gelatin, from porcine skinSIGMA-ALDRICHCat. # G1890-100GWorking concentration
of 0.1% (prepared in distilled
water and then autoclaved)
Chemical
compound, drug
Blasticidin S HCl, powderThermoFisher ScientificCat. #R21001From a stock solution
of 10 mg/ml. Prepared in
UltraPure Distilled water
(Invitrogen Cat. # 10977–015)
Chemical
compound, drug
Puromycin DihydrochlorideThermoFisher ScientificCat. #A1113803From a stock solution of 10 mg/ml

Additional files

Supplementary file 1

Miscellaneous tables listing the following information.

Vectors for expressing PLXND1 and GIPC proteins/fragments, primers for genotyping Tg(fli1a:GAL4FF)ubs4 zebrafish, oligos for assembling DNA templates for in vitro transcription of gRNAs for zebrafish genome editing and for making lentiCRISPRv2-Blast vectors for Cas9 and gRNA coexpression for use in HUVEC, cognate sequences of WT alleles and mutant alleles generated in this study via genome editing, and primers for genotyping mutant alleles generated in this study via genome editing. Related to Figures 17, Figure 2—figure supplement 1, Figure 2—figure supplement 2, Figure 4—figure supplement 1, Figure 4—figure supplement 2, Figure 5—figure supplement 1, Figure 7—figure supplement 1 and, Figure 7—figure supplement 2.

https://doi.org/10.7554/eLife.30454.021
Supplementary file 2

Tables of the raw and average densitometry values of tagged proteins in Western blots of CoIP experiments and their statistical significances.

Related to Figure 1.

https://doi.org/10.7554/eLife.30454.022
Supplementary file 3

Tables of the plxnd1skt6 complementation of plxnd1fov01b (related to Figure 2C–E), the comparison of the vascular phenotypes of homozygous WT and homozygous plxnd1skt6 mutant siblings (related to Figure 2F–I, Figure 2—figure supplement 1), and the mosaic transgenic endothelial expression of tagged forms of zebrafish Plxnd1 in plxnd1fov01b null mutants (related to Figure 2—figure supplement 2J).

https://doi.org/10.7554/eLife.30454.023
Supplementary file 4

Tables comparing the Se-DLAV truncations of wild-type embryos and plxnd1skt6 mutants (at 32 hpf) in animals treated with DMSO and SU5416. Related to Figure 3E and Figure 3—figure supplement 1.

https://doi.org/10.7554/eLife.30454.024
Supplementary file 5

Tables comparing the Se truncations of wild-type embryos and gipc mutants at 32 hpf.

https://doi.org/10.7554/eLife.30454.025
Supplementary file 6

Tables comparing the Se-DLAV truncations of gipc1skt1 (MZ) and gipc1skt1 (MZ); plxnd1fov01/+ mutants at 32 hpf.

https://doi.org/10.7554/eLife.30454.026
Supplementary file 7

Tables of raw and average densitometry values for both pERK and ERKTotal, relative ERK activities and the statistical significances of the latter. Related to Figure 7E and Figure 7—figure supplement 1.

https://doi.org/10.7554/eLife.30454.027
Supplementary file 8

Protein sequences. Related to Figure 1, Figure 2A–B, Figure 4—figure supplement 1, Figure 7—figure supplement 2, Supplementary file 1 (see ‘Vectors for expressing PLXND1 and GIPC proteins/fragments’ and ‘Cognate sequences of WT alleles and mutant alleles generated in this study via genome editing’), and Supplementary file 2.

https://doi.org/10.7554/eLife.30454.028
Transparent reporting form
https://doi.org/10.7554/eLife.30454.029

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