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Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart

Vermot, Julien and Forouhar, Arian S. and Liebling, Michael and Wu, David and Plummer, Diane and Gharib, Morteza and Fraser, Scott E. (2009) Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart. PLoS Biology, 7 (11). Art. No. e1000246. ISSN 1544-9173. PMCID PMC2773122. https://resolver.caltech.edu/CaltechAUTHORS:20091208-134946591

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Image (TIFF) (Oscillatory flow is observed in the AV canal before valves become functional.) - Supplemental Material
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Image (TIFF) (klf2a expression is localized to the endothelial cells of the AV canal.) - Supplemental Material
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Image (TIFF) (Decreased blood cells number do not affects heart chamber patterning as well as head and trunk vasculogenesis.) - Supplemental Material
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Image (TIFF) (Quantitative analysis of the blood flow observed in the AV canal at 48 hpf.) - Supplemental Material
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Image (TIFF) (Decreased retrograde flow via changes in contractility affects valve morphogenesis.) - Supplemental Material
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Image (TIFF) (Strong phenotype triggered by lidocaine treatment. ) - Supplemental Material
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Image (TIFF) (notch1b expression after lidocaine treatment.) - Supplemental Material
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Image (TIFF) (Expression of notch1b, bmp4, and cmlc2 in control ([A, C, and E], respectively) and klf2a MO-treated ([B, D, and F], respectively) embryos.) - Supplemental Material
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Image (TIFF) (Expression of three marker genes at 36 hpf in the heart of normal and klf2a morphants. ) - Supplemental Material
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Image (TIFF) (Validation of the MO strategy.) - Supplemental Material
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Image (TIFF) ((A–E) Injection of klf2a mismatch morpholino does not affect valve invagination and cell shape.) - Supplemental Material
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[img] Video (QuickTime) (Transvalvular flow changes dramatically during cardiac morphogenesis.) - Supplemental Material
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[img] Video (QuickTime) (Three dimensional reconstruction of klf2a expression in the AV canal endothelium.) - Supplemental Material
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[img] Video (QuickTime) (Transvalvular flow changes in the AV canal and atrium at 36 hpf, 48 hpf, and 56 hpf in wild-type embryos (which also serve as controls [CTL]).) - Supplemental Material
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[img] Video (QuickTime) (Transvalvular flow changes in the A–V canal in gata1 morphants, gata2 morphants and gata1/2 morphants at 48 hpf.) - Supplemental Material
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[img] Video (QuickTime) (Hematocrit is severely reduced in gata2 morphants at 48 hpf.) - Supplemental Material
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[img] Video (QuickTime) (Transvalvular flow changes in the AV canal in cx36.7, ttna, and myh6 morphants at 48 hpf.) - Supplemental Material
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[img] Video (QuickTime) (Transvalvular flow changes in the AV canal of control embryos at 3 Hz, 2.4 Hz, 1.5 Hz, and 1.2 Hz. ) - Supplemental Material
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[img] Video (QuickTime) (Reduced oscillatory flow leads to heart valve dysgenesis.) - Supplemental Material
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[img] Video (QuickTime) (Three-dimensional cell shape in the AV canal of transgenic Tg(flk1:EGFP) embryos at 72 hpf in a control embryo, in gata1, gata2, and klf2a morphants, and in a lidocaine-treated embryo.) - Supplemental Material
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Abstract

Heart valve anomalies are some of the most common congenital heart defects, yet neither the genetic nor the epigenetic forces guiding heart valve development are well understood. When functioning normally, mature heart valves prevent intracardiac retrograde blood flow; before valves develop, there is considerable regurgitation, resulting in reversing (or oscillatory) flows between the atrium and ventricle. As reversing flows are particularly strong stimuli to endothelial cells in culture, an attractive hypothesis is that heart valves form as a developmental response to retrograde blood flows through the maturing heart. Here, we exploit the relationship between oscillatory flow and heart rate to manipulate the amount of retrograde flow in the atrioventricular (AV) canal before and during valvulogenesis, and find that this leads to arrested valve growth. Using this manipulation, we determined that klf2a is normally expressed in the valve precursors in response to reversing flows, and is dramatically reduced by treatments that decrease such flows. Experimentally knocking down the expression of this shear-responsive gene with morpholine antisense oligonucleotides (MOs) results in dysfunctional valves. Thus, klf2a expression appears to be necessary for normal valve formation. This, together with its dependence on intracardiac hemodynamic forces, makes klf2a expression an early and reliable indicator of proper valve development. Together, these results demonstrate a critical role for reversing flows during valvulogenesis and show how relatively subtle perturbations of normal hemodynamic patterns can lead to both major alterations in gene expression and severe valve dysgenesis.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1371/journal.pbio.1000246DOIArticle
http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1000246PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773122/PubMed CentralArticle
ORCID:
AuthorORCID
Gharib, Morteza0000-0002-2204-9302
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2009 Vermot et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received April 1, 2009; accepted October 9, 2009; published November 17, 2009. Academic Editor: Hiroshi Hamada, Osaka University, Japan. We are grateful to L. Trinh for sharing reagents and for providing probes, M. Lardelli, T. Zhong, and H. Clevers for ISH probes, the members of the Beckman Biological Imaging Center for discussions, the Bronner-Fraser laboratory for sharing tools and reagents, and Shigehisa Hirose for providing an aliquot of cx36.7 morpholino. Support received through a National Institutes of Health (NIH) grant to SEF (P01HD037105). JV was supported by a fellowship from the Human Frontier Science Program (HFSP), AF by a fellowship from the NIH, ML by a fellowship from the Swiss National Science Foundation (PA002-111433), DW by the NIH Medical Scientist Training Program at UCLA/Caltech, and DP by a fellowship from the Summer Undergraduate Research Fellowship (SURF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Contributions: The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: JV ASF ML MG SEF. Performed the experiments: JV ASF DW DP. Analyzed the data: JV ASF ML DW MG SEF. Contributed reagents/materials/analysis tools: DW. Wrote the paper: JV ASF ML MG SEF. Movie crafting: JV ASF ML.
Group:GALCIT
Funders:
Funding AgencyGrant Number
NIHP01HD037105
Human Frontier Science ProgramUNSPECIFIED
Swiss National Science Foundation (SNSF)PA002-111433
Caltech Summer Undergraduate Research Fellowship (SURF)UNSPECIFIED
Subject Keywords:AV, atrioventricular; hpf, hours postfertilization; ISH, in situ hybridization; MO, morpholine oligonucleotide; RFF, retrograde flow fraction; WSS, wall shear stress
Issue or Number:11
PubMed Central ID:PMC2773122
Record Number:CaltechAUTHORS:20091208-134946591
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20091208-134946591
Official Citation:Vermot J, Forouhar AS, Liebling M, Wu D, Plummer D, et al. 2009 Reversing Blood Flows Act through klf2a to Ensure Normal Valvulogenesis in the Developing Heart. PLoS Biol 7(11): e1000246. doi:10.1371/journal.pbio.1000246
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:16906
Collection:CaltechAUTHORS
Deposited By: Jason Perez
Deposited On:14 Dec 2009 21:43
Last Modified:03 Oct 2019 01:19

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