Transcriptional control of cardiac neural crest cells condensation and outflow tract septation by the Smad1/5/8 inhibitor Dullard
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.
Abstract: 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.
Summary (2 min read)
- 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).
- 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.
- 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.
- 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.
- 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).
Did you find this useful? Give us your feedback
"Transcriptional control of cardiac ..." refers methods in this paper
...Immunostainings were acquired using a Nikon Ti2 microscope, driven by Metamorph (Molecular Devices), equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometrics), then assembled and analyzed on Fiji (Schindelin et al., 2012)....
..., 1998), MGI:2386570), Rosa26mTmG ((Muzumdar et al., 2007), MGI: 3716464), and C57BL/6JRj (Janvier Labs)....
...…following papers with their MGI IDs: Dullardflox/flox ((Sakaguchi et al. 2013); in these mice exons 2 to 4 are floxed), Pax3Cre ((Engleka et al., 2005), MGI: 3573783), Wnt1Cre ((Danielian et al., 1998), MGI:2386570), Rosa26mTmG ((Muzumdar et al., 2007), MGI: 3716464), and C57BL/6JRj (Janvier Labs)....
...In contrast, Pax3Cre or Wnt1Cre; Dullardflox/flox embryos exhibited asymmetric breakdown of the endocardium on the pulmonary side with obstruction of the pulmonary artery (Pa) (Figure 2Aiv - Figure supplement 2E)....
...5 Wnt1Cre; Dullardflox/+ and Wnt1Cre; Dullardflox/flox embryos....
...In recombined Wnt1Cre; Dullardflox/flox; Rosa26mTmG, the cardiac NCC displayed a strong reduction in Dullard levels, while the surrounding tissues remained Dullard positive....
...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)....
"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)....
Related Papers (5)
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.