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Transcriptional control of cardiac neural crest cells condensation and outflow tract septation by the Smad1/5/8 inhibitor Dullard

08 Aug 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 548511

TL;DR: It is shown that NCC aggregation starts more pronounced at distal OFT areas than at proximal sites, and Dullard mediated fine-tuning of BMP signalling ensures the timed and progressive condensation of NCC and rules a zipper-like closure of the OFT.

AbstractSummary The establishment of separated pulmonary and systemic circulations in vertebrates, via the cardiac outflow tract (OFT) septation, stands as a sensitive developmental process accounting for 10% of all congenital anomalies. It relies on the Neural Crest Cells (NCC) colonization of the heart, whose condensation along the endocardial wall forces its scission into two tubes. Here, we show that NCC aggregation starts more pronounced at distal OFT areas than at proximal sites. This spatial organisation correlates with a decreasing distal-proximal gradient of BMP signalling. Dullard, a nuclear phosphatase, sets the BMP gradient amplitude and prevents NCC premature condensation. Dullard is required for maintaining transcriptional programmes providing NCC with mesenchymal traits. It attenuates the adhesive cue Sema3c levels and conversely promotes the epithelial-mesenchymal transition driver Twist1 expression. Altogether, Dullard mediated fine-tuning of BMP signalling ensures the timed and progressive condensation of NCC and rules a zipper-like closure of the OFT.

Summary (2 min read)

Introduction

  • The heart outflow tract (OFT) is an embryonic structure which ensures the connection between the muscular heart chambers and the embryonic vascular network.
  • Little is known on the cardiac NCC behaviour and molecular cascades triggered by BMP signalling and responsible for the cardiac NCC mediated OFT septation.
  • Several pieces of evidence collected in drosophila, xenopus, and mouse embryos indicate that this enzyme dampens the Smad1/5/8 phosphorylation levels upon BMP stimulation (Sakaguchi et al., 2013; Satow et al., 2006; Urrutia et al., 2016).

Results

  • Dullard deletion triggers hyper-activation of BMP intracellular signalling in cardiac NCC Immuno-labelling for PSmads and GFP, and DAPI staining on transverse sections across the OFT at 3 distinct distal-proximal levels in E11.5 embryos with the indicated genotype.
  • 3D imaging of these cells thanks to the GFP reporter revealed that cardiac NCC reached similar OFT levels in E11.5 control and Dullard mutants, showing that Dullard is not required for NCC colonization of the OFT .
  • Similarly, the position of NCC to the endocardium was variable along the OFT axis of control embryos; NCC were closer to this epithelium at distal levels than at proximal levels .
  • Five sub-populations of cells could be identified based on their gene expression signature (Sub Pop1 to 5) , each of them containing an unbalanced ratio of mutant versus control cells , suggesting that Dullard influences the fate of all NCC subtypes.

Discussion

  • By investigating the function of the Dullard phosphatase during cardiac NCC-mediated OFT septation, the authors have uncovered that the BMP-dependent condensation of the cardiac NCC is spatially regulated and sets the timing of cardiac cushions fusion.
  • Prominent expression of Smad6, a BMP negative feedback effector, and of the diffusible inhibitor Noggin, are indeed observed in the OFT from E10.5 throughout great arteries formation and thus represent promising candidates (Choi, Stottmann, Yang, Meyers, & Klingensmith, 2007; Galvin et al., 2000).
  • This suggests that the regulatory influence of BMP signaling on Sema3c expression is not restricted to the context of cardiac NCC but also to other cell types.
  • The Smads direct action on Sema3c expression remains unclear.
  • The authors have uncovered part of the cellular and molecular mechanisms by which BMP controls cardiac NCC behaviour.

Experimental procedures

  • All animal experiments were approved by the Animal Ethics Committee of Sorbonne University.
  • In situ hybridization on cryosections were processed following the protocol described in (Chotteau-Lelièvre, Dollé, & Gofflot, 2006).
  • Cells were centrifuged and resuspended in the antibody solution for a 25min incubation period (4°C, dark), washed 3 times, filtered (Fisher cell strainer, 70μm mesh) and 7AAD PE-Cy7 (1/800) was added in the cells suspension to exclude dead cells.
  • Pre-amplified samples were diluted 5x with low EDTA TE buffer prior to qPCR analysis using 48.48 Dynamic Array™ IFCs and the BioMark TM HD System .
  • For unsupervised clustering, the authors used PhenoGraph that takes as input a matrix of N singlecell measurements and partitions them into subpopulations by clustering a graph that represents their phenotypic similarity.

Acknowledgments

  • The authors thank the Cadot, Bitoun and Ribes Laboratories for discussions, Sigolène Meilhac for her help on understanding heart development concepts, Edgar Gomes laboratories, Isabelle Le Roux and Pascale Gilhardi-Hebenstreit for useful comments, Stéphane Zaffran for the Sema3c ISH probe and Morgane Belle for Lightsheet microscopy.
  • The Pax3Cre and Rosa26mTmG transgenic lines were kindly provided by F. Relaix, and the Wnt1Cre line by A. Pierani.
  • This work was supported by Agence Nationale pour la Recherche (ANR-14CE09-0006-04) to BC; Association Institut de Myologie to BC.
  • MV has a postdoctoral fellowship from the Laboratoire d'Excellence Revive (Investissement d'Avenir; ANR-10-LABX-73).

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1
Transcriptional control of cardiac neural crest cells condensation and outflow
tract septation by the Smad1/5/8 inhibitor Dullard
Jean-François Darrigrand
1
, Mariana Valente
2
, Pauline Martinez
1
, Glenda Comai
3
, Maxime Petit
4
,
Ryuichi Nishinakamura
5
, Daniel S. Osorio
6
, Vanessa Ribes
7#
, Bruno Cadot
1#
.
1. INSERM - Sorbonne Université UMR974 - Center for Research in Myology, 105 boulevard de l'Hôpital,
75634 Paris Cedex 13, France.
2. Cellular, Molecular, and Physiological Mechanisms of Heart Failure team, Paris-Cardiovascular Research
Center (PARCC), European Georges Pompidou Hospital (HEGP), INSERM U970, F-75737 Paris Cedex 15,
France.
3. Stem Cells and Development, Department of Developmental & Stem Cell Biology, CNRS UMR 3738,
Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.
4. Unité Lymphopoïèse INSERM U1223, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France.
5. Institute of Molecular Embryology and Genetics, Kumamoto University, Japan.
6. Cytoskeletal Dynamics Lab, Institute for Molecular and Cellular Biology, Instituto de Investigação e
Inovação em Saúde, Universidade do Porto, Portugal.
7. Institut Jacques Monod, CNRS UMR7592, Université Paris Diderot, Sorbonne Paris Cité, 75205 Paris
Cedex, France.
# corresponding authors: vanessa.ribes@ijm.fr, bruno.cadot@inserm.fr
lead contact: bruno.cadot@inserm.fr
Summary
The establishment of separated pulmonary and systemic circulations in vertebrates, via the cardiac
outflow tract (OFT) septation, stands as a sensitive developmental process accounting for 10% of all
congenital anomalies. It relies on the Neural Crest Cells (NCC) colonization of the heart, whose
condensation along the endocardial wall forces its scission into two tubes. Here, we show that NCC
aggregation starts more pronounced at distal OFT areas than at proximal sites. This spatial
organisation correlates with a decreasing distal-proximal gradient of BMP signalling. Dullard, a nuclear
phosphatase, sets the BMP gradient amplitude and prevents NCC premature condensation. Dullard is
required for maintaining transcriptional programmes providing NCC with mesenchymal traits. It
attenuates the adhesive cue Sema3c levels and conversely promotes the epithelial-mesenchymal
transition driver Twist1 expression. Altogether, Dullard mediated fine-tuning of BMP signalling
ensures the timed and progressive condensation of NCC and rules a zipper-like closure of the OFT.
Keywords
Outflow tract, Neural crest cells, Dullard, BMP signalling, Mesenchymal-epithelial transition
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted August 8, 2019. ; https://doi.org/10.1101/548511doi: bioRxiv preprint

1
Introduction
The heart outflow tract (OFT) is an embryonic structure
which ensures the connection between the muscular heart
chambers and the embryonic vascular network. Initially
forming a solitary tube called truncus arteriosus, it gets
progressively remodelled into two tubes which will give rise
to the aortic (Ao) and pulmonary (Pa) arteries ((Brickner,
Hillis, & Lange, 2000); Figure 1A). This remodelling stands as
one of the most sensitive morphogenesis processes. As
such, faulty septation of the OFT represents 30% of all
congenital heart diseases, with poor clinical prognosis due
to improper mixing of oxygenated and deoxygenated blood.
This thus calls for a better understanding of the cellular and
molecular cues by which the OFT gets septated during
development.
The morphogenesis of the OFT is orchestrated in time and
space by several cross-interacting cell-types including the
myocardial progenitors of the second heart field (SHF), the
endocardial cells (EC) delineating the OFT lumen, and the
cardiac neural crest cells (cardiac NCC) (Kelly, 2012; Keyte &
Hutson, 2012) (Figure 1A). Various genetic manipulations or
ablation models have highlighted the predominant role of
cardiac NCC in initiating and controlling OFT septation
(Bockman, Redmond, Waldo, Davis, & Kirby, 1987; Phillips et
al., 2013). Originally, cardiac NCC delaminate from the
dorsal neural tube and migrate through the pharyngeal
mesoderm to reach the developing OFT (Figure 1A). There,
they invade the two cardiac cushions, condense towards the
endocardium and trigger its rupture, thereby inducing
cardiac cushions fusion and creating the two great arteries
(Plein et al., 2015; Waldo, Miyagawa-Tomita, Kumiski, &
Kirby, 1998). The rupture of the endocardium is first
detected in the regions of the OFT which are the most distal
from the heart chambers. In mouse embryos this rupture
occurs around 11.5 days of embryonic development (E11.5;
Figure 1A) and then expands progressively to more proximal
levels. In parallel to these morphogenetic events, NCC will
differentiate into the vascular smooth muscles of the aortic
arch (Keyte & Hutson, 2012). In addition, NCC will also
contribute to the arterial valves (Odelin et al., 2018).
The intense investigations to identify the molecular cues
controlling cardiac NCC stereotyped behaviour and
differentiation in the OFT mesenchyme have established the
importance of the Bone Morphogenic Proteins (BMP),
secreted by the outlying myocardium cells from E8.75
onwards (Danesh, Villasenor, Chong, Soukup, & Cleaver,
2009; Jiao et al., 2003; Liu et al., 2004; McCulley, Kang,
Martin, & Black, 2008). Indeed, the ablation of Bmpr1a, one
of their receptors, or of Smad4 a key downstream
transcriptional effectors or conversely the forced expression
of Smad7 a BMP signalling antagonist within the NCC lineage
all lead to the formation of hypoplastic cushions, a shorter
and non-septated OFT, thus phenocopying cardiac NCC
ablation experiments (Jia et al., 2007; Stottmann, Choi,
Mishina, Meyers, & Klingensmith, 2004; Tang, Snider, Firulli,
& Conway, 2010). The knock-out of the ligand BMP4 from
the myocardium similarly prevents OFT septation (Liu et al.,
2004). However, little is known on the cardiac NCC
behaviour and molecular cascades triggered by BMP
signalling and responsible for the cardiac NCC mediated OFT
septation.
To get insight into these molecular cascades, we decided to
dissect the role of Dullard (Ctdnep1) during OFT
morphogenesis, a perinuclear phosphatase uncovered as a
potential BMP intracellular signalling inhibitor (Sakaguchi et
al., 2013; Urrutia, Aleman, & Eivers, 2016). In the canonical
BMP signalling cascade, the binding of BMP ligands to their
transmembrane receptors leads to the phosphorylation of
the transcription factors Smad1/5/8, which translocate to
the nucleus and modify the transcriptional landscape of
targeted cells (Bruce & Sapkota, 2012). Out of the few
cytoplasmic modulators of this phosphorylation identified,
including PP1A, PP2B, the inhibitory Smads 6 and 7 and the
Ubiquitin degradation pathway, stands Dullard (Bruce &
Sapkota, 2012). The Dullard protein is evolutionary
conserved from yeast to mammals and expressed in many
embryonic tissues, including the developing neural tube and
neural crest cells (Sakaguchi et al., 2013; Satow, Kurisaki,
Chan, Hamazaki, & Asashima, 2006; Tanaka et al., 2013;
Urrutia et al., 2016)(Figure 1D). Several pieces of evidence
collected in drosophila, xenopus, and mouse embryos
indicate that this enzyme dampens the Smad1/5/8
phosphorylation levels upon BMP stimulation (Sakaguchi et
al., 2013; Satow et al., 2006; Urrutia et al., 2016). However,
this activity is likely to be tissue specific, as depleting Dullard
in the early mouse embryos does not impair BMP signalling,
while its depletion in the mouse embryonic kidneys leads to
elevated response of cells to BMPs (Sakaguchi et al., 2013;
Tanaka et al., 2013). It is worth noting that in these two
cases, Dullard appeared as a key regulator of the
morphogenetic events regulating the elaboration of
embryonic tissues. While it is required early in development
for the expansion of extraembryonic tissues, later on it
prevents cell death by apoptosis in kidney nephrons
(Sakaguchi et al., 2013; Tanaka et al., 2013).
We showed here that deletion of Dullard in the cardiac NCC
increases Smad1/5/8 activity, leading to premature and
asymmetric septation of the OFT and pulmonary artery
closure. This BMP overactivation in the cardiac NCC triggers
the downregulation of mesenchymal markers (Snai2,
Twist1, Rac1, Mmp14 and Cdh2) and the upregulation of
Sema3c, associated with premature cardiac NCC
condensation to the endocardium. Our data converge to a
model whereby graded BMP activity, Sema3c expression
and cardiac NCC condensation along the OFT axis set the
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted August 8, 2019. ; https://doi.org/10.1101/548511doi: bioRxiv preprint

2
tempo of its septation from its distal to its proximal regions.
Hence, our findings reveal that fine tuning of BMP signalling
levels in cardiac NCC orchestrates OFT septation in time and
space.
Results
Dullard deletion triggers hyper-activation of BMP
intracellular signalling in cardiac NCC
In order to ablate Dullard in cardiac NCC, we crossed mice
carrying floxed alleles of Dullard with mice expressing the
Cre recombinase from the Pax3 or Wnt1 loci (Danielian,
Muccino, Rowitch, Michael, & McMahon, 1998; Engleka et
al., 2005; Sakaguchi et al., 2013). Cell lineage tracing was
achieved by using a ubiquitous double-fluorescent Cre
reporter allele, Rosa26mTmG, in which Cre-mediated
recombination labels the cells with membrane-targeted GFP
(Muzumdar, Tasic, Miyamichi, Li, & Luo, 2007). The pattern
of cell recombination in the cardiac cushions of E11.5
control embryos carrying either Cre drivers matched with
the pattern of colonizing cardiac NCC described by previous
lineage analyses (Figure 1B) (Brown et al., 2001; Jiang,
Rowitch, Soriano, McMahon, & Sucov, 2000). RT-qPCR on
single Fac-sorted (FACS) cells from
dissected OFT and
RNAscope in situ hybridization on histological sections were
used to monitor Dullard expression (Figure 1C,D). At E11.5,
Dullard was ubiquitously expressed in all OFT layers of
control embryos. In recombined Wnt1
Cre
; Dullard
flox/flox
;
Rosa26mTmG, the cardiac NCC displayed a strong reduction
in Dullard levels, while the surrounding tissues remained
Dullard positive. Strikingly, in these mutants, the NCC
formed a unique mass at the distal part of the OFT, while
two distinct NCC cushions were present in the control
embryos (Figure 1D,E, 2A,B), indicating that Dullard
regulates the spatial organization of NCC in the OFT (see
below).
We next assessed the relationship between Dullard and the
activity of the intracellular effector of BMP signalling, i.e. the
Smad1/5/8 (Smads) transcription factors, in mammalian
cells. As previously shown (Satow et al., 2006) in the
myogenic cell line C2C12, overexpression of Dullard strongly
decreased the levels of phosphorylated Smads (PSmads)
induced by BMP2 treatment (Figure supplement 1A). In
addition, by generating a version of Dullard carrying a
phosphatase dead domain, we could show that this
inhibitory role relied on Dullard phosphatase activity (Figure
supplement 1A). In cardiac NCC, the deletion of Dullard was
sufficient to double the levels of PSmads within these cells,
whatever their position along the distal-proximal OFT axis of
E11.5 hearts (Figure 1E - Figure supplement 1B). Hence in
this embryonic cell lineage, Dullard also acts as a BMP
intracellular signalling inhibitor. Interestingly, in both
control and mutant contexts, PSmads levels were more
elevated distally than proximally (Figure 1Eiii - Figure
supplement 1B), indicating that cardiac NCC harbour a
graded BMP response, which declines as they colonize more
proximal OFT areas. Dullard in cardiac NCC is required to
dampen the magnitude of the BMP signalling response
gradient along the OFT length, but does not control its
establishment (Figure 1Eiii).
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The copyright holder for this preprint (which wasthis version posted August 8, 2019. ; https://doi.org/10.1101/548511doi: bioRxiv preprint

3
Figure 1: Dullard acts as a Smad1/5/8 activity inhibitor in cardiac NCC.
(A) Ai Schematic representation of the migration routes the cardiac NCC
(green) have taken to reach the heart region (red) in a E10.5 mouse embryo.
Aii Schematics of the embryonic heart at E11.5 showing the distal-proximal
axis of the OFT. Aiii Schematic representation of transverse sections through
the OFT showing discrete stages of NCC condensation and endocardium
septation along the OFT distal-proximal axis. (B) Pecam and GFP immuno-
labelling and DAPI staining on transverse sections throughout the medial OFT
of E11.5 Wnt1
Cre
or Pax3
Cre
; Dullard
flox/+
; Rosa26mTmG embryos. (C)
Normalized expression levels of Dullard assayed by q-RT-PCR on single cells
isolated from E11.5 Wnt1
Cre
; Dullard
flox/+
and Wnt1
Cre
; Dullard
flox/flox
;
Rosa26mTmG hearts (dots: value for a single cell; boxplot: mean± s.e.m.). The
probe monitoring Dullard expression specifically binds to exons 2-3, which are
the exons recombined by the Cre recombinase. (D) Dullard mRNA distribution
detected using RNAscope probes, in transverse sections of E11.5 control and
mutant OFTs, assessed by RNAscope. Dullard mRNA levels were significantly
reduced in mutant cardiac cushions compared to controls; however, mRNA
signals were still detected given the binding of Z pair probes to non-
recombined exons 5 to 8 and UTR region. (E) Ei. Schematics of E11.5 heart
showing the position of the transverse sections used to quantify the levels of
the phosphorylated forms of Smad1/5/8 (PSmads) in iii. Eii. Immuno-labelling
for PSmads and GFP, and DAPI staining on transverse sections across the OFT
at 3 distinct distal-proximal levels in E11.5 embryos with the indicated
genotype. Pale green dotted lines delineate the area colonized by cardiac NCC.
Eiii. Quantification of PSmads levels in cardiac NCC along the OFT distal-
proximal axis of E11.5 embryos with the indicated genotype (dots: values
obtained on a given section; n>4 embryos per genotype recovered from at
least 3 liters; the black line is the linear regression, the coloured areas
delineate the 95% confidence intervals, ***: p-value < 0,001 for a two way-
Anova statistical test). Ao: aortic artery, Pa: pulmonary artery.
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not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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4
Dullard is a key modulator of cardiac NCC mediated OFT
septation
We next sought to examine the morphology of the OFT in
control and Dullard mutants, knowing that cardiac NCC
controls its septation (Bockman et al., 1987; Phillips et al.,
2013; Plein et al., 2015). For this, we monitored, using 3D
lightsheet and confocal microscopy, E11.5-12 hearts
labelled for the arterial marker, Pecam (Figure 2A,B - Figure
supplement 2E, Movie supplement 1, 2). At distal levels, the
OFT of control embryos displayed symmetrical septation
with two great arteries of similar size (Figure 2Ai). In
contrast, Pax3
Cre
or Wnt1
Cre
; Dullard
flox/flox
embryos
exhibited asymmetric breakdown of the endocardium on
the pulmonary side with obstruction of the pulmonary
artery (Pa) (Figure 2Aiv - Figure supplement 2E). At more
medial levels in control embryos, the aortic and pulmonary
endocardium regions were connected and attached to the
presumptive pulmonary valve intercalated-cushion (PV-IC)
(Figure 2Aii,iii). Conversely in mutants, this attachment was
absent, and the endocardium was displaced towards the
aortic side, highlighting premature cushions fusion and OFT
septation (Figure 2Av, vi - Figure supplement 2E).
To dig into the cellular mechanisms by which Dullard
prevents the appearance of these heart defects, we
evaluated the migrative, proliferation and death status of
cardiac NCC. 3D imaging of these cells thanks to the GFP
reporter revealed that cardiac NCC reached similar OFT
levels in E11.5 control and Dullard mutants, showing that
Dullard is not required for NCC colonization of the OFT
(Figure 2C - Figure supplement 2A, Movie supplement 2-6).
Similarly, staining and quantifying cells in mitosis or in
apoptosis using antibodies raised against the
phosphorylated form of histone H3 and the cleaved version
of Caspase 3, respectively, indicated that Dullard does not
control the amplification nor the survival of cardiac NCC
(Figure supplement 2B,C). In agreement with these
observations, the total number of GFP
+
cells colonizing the
OFT in mutant embryos was not significantly different from
that found in controls (Figure 2D).
Finally, we wondered whether the morphogenetic defects of
the mutant OFT could stem from differences in cell-cell
arrangements, looking at the position and orientation of
DAPI labelled NCC and endocardium nuclei (Figure 2Ai’-vi’,E-
G). Quantification of the shortest distance between cardiac
NCC nuclei indicated that in E11.5 control hearts, NCC
condensation was variable along the distal-proximal axis of
the OFT (Figure 2E,F). Cells were closer to each other at
distal levels than in proximal regions. This progression of
NCC condensation along the OFT axis was impaired in
Dullard mutants. Mutant NCC prematurely condensed
notably within the medial region of the OFT (Figure 2E,F).
Similarly, the position of NCC to the endocardium was
variable along the OFT axis of control embryos; NCC were
closer to this epithelium at distal levels than at proximal
levels (Figure 2E, G). In mutants, the NCC were in a closer
vicinity of the endocardium than control cells, so that in
medial levels they displayed traits of cells normally found at
distal levels in control hearts (Figure 2E,G). In an agreement
with these data, the OFT area was reduced in mutants and
remained constant along the distal to proximal axis (Figure
supplement 2D). Finally, the orientation of the cardiac NCC
nuclei relative to the endocardium appeared also spatially
regulated along the proximal-distal axis of the OFT, in both
mutant and control hearts (Figure 2Ai’-vi’,E - Figure
supplement 2E). In controls, at distal levels NCC were
perpendicular to the endocardium, while at proximal levels
no orientation preference could be assigned (Figure 2Ai’-ii’,
E, Figure supplement 2E). Strikingly, in Wnt1
Cre
; Dullard
flox/flox
OFTs the perpendicular orientation was more widely
observed than in control OFT (Figure 2Aiv’-vi’,E - Figure
supplement 2E).
Altogether these data demonstrate that Dullard stands as a
key modulator of NCC behavior dynamics in the heart
and hence of OFT septation. It hampers NCC condensation,
and thereby leads to the premature breakage of the
endocardium and to obstruction of the pulmonary artery.
.CC-BY-NC-ND 4.0 International licenseavailable under a
not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (which wasthis version posted August 8, 2019. ; https://doi.org/10.1101/548511doi: bioRxiv preprint

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  • ...5 Wnt1Cre; Dullardflox/+ and Wnt1Cre; Dullardflox/flox; Rosa26mTmG hearts (dots: value for a single cell; boxplot: mean± s.e.m.)....

    [...]

  • ...Dullard deletion triggers hyper-activation of BMP intracellular signalling in cardiac NCC In order to ablate Dullard in cardiac NCC, we crossed mice carrying floxed alleles of Dullard with mice expressing the Cre recombinase from the Pax3 or Wnt1 loci (Danielian, Muccino, Rowitch, Michael, & McMahon, 1998; Engleka et al., 2005; Sakaguchi et al., 2013)....

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Journal ArticleDOI
TL;DR: In adults, the most common causes of cyanotic congenital heart disease are tetralogy of Fallot61 and Eisenmenger's syndrome.
Abstract: Cyanotic Conditions Patients with cyanotic congenital heart disease have arterial oxygen desaturation resulting from the shunting of systemic venous blood to the arterial circulation. The magnitude of shunting determines the severity of desaturation. Most children with cyanotic heart disease do not survive to adulthood without surgical intervention. In adults, the most common causes of cyanotic congenital heart disease are tetralogy of Fallot61 and Eisenmenger's syndrome. Tetralogy of Fallot Tetralogy of Fallot, the most common cyanotic congenital heart defect after infancy, is characterized by a large ventricular septal defect, an aorta that overrides the left and right ventricles, obstruction of the . . .

790 citations


Journal ArticleDOI
TL;DR: The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants, and defects highlight the importance of Smads in the tissue-specific modulation of Tgf-β superfamily signalling pathways in vivo.
Abstract: Smad proteins are intracellular mediators of signalling initiated by Tgf-βsuperfamily ligands (Tgf-βs, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses1. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-β superfamily ligands, and downregulate signalling in in vitro assays2,3,4,5,6,7. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-β superfamily signalling pathways in vivo.

449 citations


"Transcriptional control of cardiac ..." refers background in this paper

  • ...5 throughout great arteries formation and thus represent promising candidates (Choi, Stottmann, Yang, Meyers, & Klingensmith, 2007; Galvin et al., 2000)....

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Frequently Asked Questions (1)
Q1. What have the authors contributed in "Transcriptional control of cardiac neural crest cells condensation and outflow tract septation by the smad1/5/8 inhibitor dullard" ?

In this paper, the authors identify the molecular cues controlling cardiac NCC stereotyped behaviour and differentiation in the OFT mesenchyme.