Venous endothelin modulates responsiveness of cardiac sympathetic axons to arterial semaphorin

  1. Denise M Poltavski
  2. Pauline Colombier
  3. Jianxin Hu
  4. Alicia Duron
  5. Brian L Black
  6. Takako Makita  Is a corresponding author
  1. Children’s Hospital Los Angeles, University of Southern California Keck School of Medicine, United States
  2. University of California, San Francisco, United States
  3. Medical University of South Carolina, United States
9 figures, 1 table and 1 additional file

Figures

Edn1-Ednra signaling mutant STGs project ectopically.

(a–h) Wholemount Th immunostaining visualizing sympathetic projections from STGs in E15.5 thoraxes (a–d) and hearts (e–h) from Ednra-/- (b, f), Edn1-/- (c, g), Ece1-/- (d, h) and a control (a, e) …

https://doi.org/10.7554/eLife.42528.002
Lack of repulsive response to arterial segments in Ednra-deficient STGs in vitro.

(a–b) Neurite outgrowths from E14.5 control STG (a) and Ednra-/- STG (b) cocultured with thoracic descending aorta segments (dAo) dissected from littermate control embryos were visualized by …

https://doi.org/10.7554/eLife.42528.003
Reduced expression of Plxna4 in Ednra-deficient STGs.

(a) Quantitative real-time PCR analysis for class three semaphorin receptor and co-receptor genes. Total RNA was extracted from individual E14.5 STG isolated from total seven different litters, …

https://doi.org/10.7554/eLife.42528.004
The Plxna4 locus contains several bona fide, conserved MEF2C sites and expression is induced by endothelin signaling in a MEF2C-dependent manner.

(a) Schematic diagram of conserved MEF2C binding sites in the Plxna4 locus. The Plxna4 locus is shown in the center with transcriptional start site (TSS, bent arrow) and exons (vertical black lines) …

https://doi.org/10.7554/eLife.42528.005
Figure 5 with 1 supplement
Ednra and Plxna4 interact genetically.

(a–d) Wholemount immunostaining visualizing the dorsal surface of hearts from a 7-week-old Th-Cre; Ednrafl/+; Plxna4+/- double heterozygote (c), a Th-Cre; Ednrafl/- mutant (d), and single …

https://doi.org/10.7554/eLife.42528.006
Figure 5—figure supplement 1
Quantitative assessment of Ednra and Plxna4 genetic interaction.

(a) A compiled representation of Th+ area on the heart dorsal surface of 6–7 week old Th-Cre; Ednrafl/+; Plxna4+/- double heterozygote (n = 7), Th-Cre; Ednrafl/- mutant (n = 4), and littermate …

https://doi.org/10.7554/eLife.42528.007
Figure 6 with 1 supplement
Ednra-Plxna4 double-heterozygous mice exhibit diminished cardiac sympathetic nerve activity.

(a–c) Changes in heart rate following amphetamine (a;<2 min, (b;>10 min) and isoproterenol (c) administration measured in indicated 7-week-old single heterozygotes, double heterozygotes, conditional …

https://doi.org/10.7554/eLife.42528.008
Figure 6—figure supplement 1
Reduced baseline sympathetic activity in Ednra-Plxna4 trans-heterozygous heart.

(a–c) Power spectrum plots of baseline heart rate fluctuation in Th-Cre; Ednrafl/+; Plxna4+/- (b), Th-Cre; Ednrafl/- (c) mutants, and a littermate control (a). The cardiac interbeat intervals in …

https://doi.org/10.7554/eLife.42528.009
Normal SCG axonal projections along the external carotid arteries in Ednra-Plxna4 trans-heterozygotes.

(a–h) Transverse sections from E14.5 Ednra+/-; Plxna4+/- (b, f), Plxna4-/- (c, g), Ednra-/- (d, h) mutant embryos and a littermate control embryo at the level of carotid artery bifurcation (a–d) and …

https://doi.org/10.7554/eLife.42528.010
Figure 8 with 1 supplement
Ednra-deficient STGs show reduced repulsive response to Sema3a in vitro.

(a–h) Neurite outgrowth from explanted STG co-cultured with COS cell aggregates were visualized by whole mount Th staining. STG dissected from E14.5 embryos of the genotypes indicated at left were …

https://doi.org/10.7554/eLife.42528.011
Figure 8—figure supplement 1
Semaphorins are expressed in arteries but not veins.

(a–h) Transverse sections from an E14.5 Ednra-/- mutant (b, f, j, n) and littermate control (a, e, i, m) embryos at the thoracic levels stained with Sema3a (a–b), Sema3f (e–f), Sema6a (i–j) and …

https://doi.org/10.7554/eLife.42528.012
A model of second-order axon guidance for cardiac sympathetic axons.

An Ednra-expressing STG neuron located in the vicinity of distinct vascular trajectories (vein and artery) sees venous-derived endothelin as a primary attractive guidance cue and initiates axonal …

https://doi.org/10.7554/eLife.42528.013

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(M. musculus)
Ednrafl, ETAfloxPMID: 14585980MGI:2687362,
RRID:IMSR_RBRC06323
Genetic reagent
(M. musculus)
MEF2Cfl, Mef2cloxPPMID: 17336904MGI:3718323
Genetic reagent
(M. musculus)
Edn1-, EdnRA-, ET-1-PMID: 8152482MGI:1857637
Genetic reagent
(M. musculus)
Ece1-, ECE-1 -PMID: 9449665MGI:2158947,
RRID:IMSR_RBRC06166
Genetic reagent
(M. musculus)
Plxna4-, PLA4 KO,
PlexinA4-
PMID: 15721238MGI:3579185,
RRID:MMRRC_030407-MU
Genetic reagent
(M. musculus)
ThCre, Th::CrePMID: 1785595MGI:5607431,
RRID:MMRRC_029177-UCD
Genetic reagent
(M. musculus)
Wnt1Cre,
Tg(Wnt1-cre/Esr1)10Rth
PMID: 9843687MGI:2447684
Cell line
(Cercopithecus aethiops)
COS-7ATCCCRL-1651,
RRID: CVCL_0224
LOT: 5784927
Originally tested
negative for Mycoplasma,
Bacterial and fungal
contamination (purchase date:6/30/2016).
Cells have not independently
authenticated for each passage used
for experiments and data collection.
Cells were not independently
tested for mycoplasma contamination.
AntibodySheep polyclonal
anti-Tyrosine
Hydroxylase
MilliporeAB1542,
RRID:AB_90755
IHC (1:400)
AntibodyRabbit polyclonal
anti-Plexin A4
Novus BiologicalMBP1-85128,
RRID:AB_11013411
IHC (1:400)
AntibodyRabbit polyclonal
anti-Semaphorin 3A
Abcamab23393,
RRID: AB_447408
IHC (1:300)
AntibodyRabbit polyclonal
anti-Semaphorin 3F
Abcaman203394,
RRID:AB_2783521
IHC (1:500)
AntibodyRabbit polyclonal
anti-Semaphorin 6A
Abcamab72369IHC (1:200)
AntibodyMouse monoclonal
anti-Semaphorin 6B
SCBTsc-390928,
RRID:AB_2783522
IHC (1:200)
AntibodyRabbit polyclonal
anti-HCN4
Abcamab69054,
RRID: AB_1861080
IHC (1:500)
AntibodyMouse monoclonal
anti-SYP
SCBTsc-17750,
RRID: AB_628311
IHC (1:500)
Recombinant
DNA reagent
Ad-GFP-2A-mSema3AVectorBioADV-271580
Sequence-based
reagent
qRT-PCR primers (hprt)primer bank
ID: 7305155a1
Sequence-based
reagent
qRT-PCR primers (nrp1)primer bank
ID: 6679134a1
Sequence-based
reagent
qRT-PCR primers (plxna3)primer bank
ID: 667939a1
Sequence-based
reagent
qRT-PCR primers (plxna4)primer bank
ID: 28461143a1
Commercial
assay or kit
RNaseOUTThermofisher10777019
Commercial
assay or kit
Trizol LSThermofisher10296028
Commercial
assay or kit
RNeasy Micro KitQiagen74004
Commercial
assay or kit
Ovation RNA-Seq
System V2
NuGEN
Commercial
assay or kit
Encore Rapid
Library System
NuGEN
Chemical
compound, drug
amphetamineSigma A5880
Chemical
compound, drug
isoproterenolSigma I6504
Chemical
compound, drug
propranololSigma P0884
Software, algorithmFiji - Image Jhttps://imagej.net/FijiRRID:SCR_003070
Software, algorithmCASAVA (v1.8.2)IlluminaRRID:SCR_001802
Software, algorithmtophat v1.3Trapnell et al. (2009)doi:10.1093/
bioinformatics/btp120
Software, algorithmU-Seq version
8.0.2
Nix et al. (2008)doi:10.1186/1471
-2105-9-523
Software, algorithmLabChart 8AD InstrumentsRRID:SCR_001620

Additional files

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