Discovery of novel determinants of endothelial lineage using chimeric heterokaryons
- Houston Methodist Research Institute, United States
- University of Iowa, United States
- Stanford University School of Medicine, United States
Abstract
We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.
Data availability
-
Discovery of Novel Determinants of Endothelial Lineage: Insights from Chimeric HeterokaryonsPublicly available at the NCBI Gene Expression Omnibus (accession no. GSE84558).
Article and author information
Author details
Funding
National Institutes of Health
- John P Cooke
American Heart Association
- Wing Tak Wong
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Gordana Vunjak-Novakovic, Columbia University, United States
Ethics
Animal experimentation: Zebrafish are kept according to the laboratory protocols described in Zebrafish: A Practical Approach (Oxford University Press, 2002). These protocols comply with the Guide for the Care and Use of Laboratory Animals, the American Association for the Accreditation of Laboratory Animal Care (AAALAC) standards, and the regulations set forth in the Animals Welfare Act (P.L. 89-544, as amended by P.L. 91-579 and P.L . 94-279). Veterinary care is provided on a 24 hours basis, including weekends and holidays, by a staff of veterinarians with specialties in laboratory animal medicine and anesthesiology, and licensed animal health technicians. Training classes are offered. All veterinary care is provided by Houston Methodist Research Institute, which is fully accredited by AAALAC (ID A4555-01) and holds an approved NIH Assurance and USDA License (start date 03/08/2013). Support includes quarantine rooms, sterile operating rooms, post-surgical recovery rooms, radiology and diagnostic laboratory services. All surgery procedures were performed under anesthesia with Tricaine 0.02 mg/ml.
Version history
- Received: November 23, 2016
- Accepted: March 17, 2017
- Accepted Manuscript published: March 21, 2017 (version 1)
- Version of Record published: April 13, 2017 (version 2)
- Version of Record updated: April 24, 2017 (version 3)
Copyright
© 2017, Wong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,452
- views
-
- 257
- downloads
-
- 7
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Discovery of novel determinants of endothelial lineage using chimeric heterokaryons
eLife 6:e23588.
https://doi.org/10.7554/eLife.23588
Further reading
-
- Cell Biology
The spindle assembly checkpoint (SAC) temporally regulates mitosis by preventing progression from metaphase to anaphase until all chromosomes are correctly attached to the mitotic spindle. Centrosomes refine the spatial organization of the mitotic spindle at the spindle poles. However, centrosome loss leads to elongated mitosis, suggesting that centrosomes also inform the temporal organization of mitosis in mammalian cells. Here, we find that the mitotic delay in acentrosomal cells is enforced by the SAC in a MPS1-dependent manner, and that a SAC-dependent mitotic delay is required for bipolar cell division to occur in acentrosomal cells. Although acentrosomal cells become polyploid, polyploidy is not sufficient to cause dependency on a SAC-mediated delay to complete cell division. Rather, the division failure in absence of MPS1 activity results from mitotic exit occurring before acentrosomal spindles can become bipolar. Furthermore, prevention of centrosome separation suffices to make cell division reliant on a SAC-dependent mitotic delay. Thus, centrosomes and their definition of two spindle poles early in mitosis provide a ‘timely two-ness’ that allows cell division to occur in absence of a SAC-dependent mitotic delay.
-
- Cell Biology
Fertilization occurs before the completion of oocyte meiosis in the majority of animal species and sperm contents move long distances within the zygotes of mouse and C. elegans. If incorporated into the meiotic spindle, paternal chromosomes could be expelled into a polar body resulting in lethal monosomy. Through live imaging of fertilization in C. elegans, we found that the microtubule disassembling enzymes, katanin and kinesin-13 limit long-range movement of sperm contents and that maternal ataxin-2 maintains paternal DNA and paternal mitochondria as a cohesive unit that moves together. Depletion of katanin or double depletion of kinesin-13 and ataxin-2 resulted in the capture of the sperm contents by the meiotic spindle. Thus limiting movement of sperm contents and maintaining cohesion of sperm contents within the zygote both contribute to preventing premature interaction between maternal and paternal genomes.
