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1.
Nat Commun ; 12(1): 5126, 2021 08 26.
Article in English | MEDLINE | ID: mdl-34446705

ABSTRACT

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.


Subject(s)
Amnion/metabolism , Macaca fascicularis/embryology , Mesoderm/embryology , Amnion/embryology , Animals , Bone Morphogenetic Protein 4/metabolism , Embryonic Development , Female , Gene Expression Regulation, Developmental , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Macaca fascicularis/genetics , Macaca fascicularis/metabolism , Mesoderm/metabolism , Pregnancy , Signal Transduction
3.
Stem Cells ; 38(10): 1267-1278, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32497389

ABSTRACT

A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.


Subject(s)
Cell Lineage , Fibroblasts/cytology , Human Embryonic Stem Cells/cytology , Myocardium/cytology , Organogenesis , Cell Differentiation , Cell Lineage/genetics , Cell Proliferation , Fetal Heart/physiology , Fibroblasts/metabolism , Humans , LIM-Homeodomain Proteins/metabolism , Muscle Development , Myocytes, Cardiac/cytology , Organogenesis/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Time Factors , Transcription Factors/metabolism , Transcription, Genetic
4.
Dev Cell ; 48(4): 475-490.e7, 2019 02 25.
Article in English | MEDLINE | ID: mdl-30713072

ABSTRACT

The morphogenetic process of mammalian cardiac development is complex and highly regulated spatiotemporally by multipotent cardiac stem/progenitor cells (CPCs). Mouse studies have been informative for understanding mammalian cardiogenesis; however, similar insights have been poorly established in humans. Here, we report comprehensive gene expression profiles of human cardiac derivatives from multipotent CPCs to intermediates and mature cardiac cells by population and single-cell RNA-seq using human embryonic stem cell-derived and embryonic/fetal heart-derived cardiac cells micro-dissected from specific heart compartments. Importantly, we discover a uniquely human subset of cono-ventricular region-specific CPCs, marked by LGR5. At 4 to 5 weeks of fetal age, the LGR5+ population appears to emerge specifically in the proximal outflow tract of human embryonic hearts and thereafter promotes cardiac development and alignment through expansion of the ISL1+TNNT2+ intermediates. The current study contributes to a deeper understanding of human cardiogenesis, which may uncover the putative origins of certain human congenital cardiac malformations.


Subject(s)
Cell Differentiation/physiology , Myocytes, Cardiac/metabolism , Receptors, G-Protein-Coupled/metabolism , Single-Cell Analysis , Animals , Cell Differentiation/genetics , Cell Line , Cells, Cultured , Embryonic Stem Cells/metabolism , Endothelial Cells/metabolism , Heart Ventricles/metabolism , Human Embryonic Stem Cells/metabolism , Humans , LIM-Homeodomain Proteins/genetics , Mice, Inbred C57BL , Multipotent Stem Cells , Myocardium/metabolism , Organogenesis , Single-Cell Analysis/methods
5.
Mol Ther ; 26(7): 1644-1659, 2018 07 05.
Article in English | MEDLINE | ID: mdl-29606507

ABSTRACT

The generation of human pluripotent stem cell (hPSC)-derived ventricular progenitors and their assembly into a 3-dimensional in vivo functional ventricular heart patch has remained an elusive goal. Herein, we report the generation of an enriched pool of hPSC-derived ventricular progenitors (HVPs), which can expand, differentiate, self-assemble, and mature into a functional ventricular patch in vivo without the aid of any gel or matrix. We documented a specific temporal window, in which the HVPs will engraft in vivo. On day 6 of differentiation, HVPs were enriched by depleting cells positive for pluripotency marker TRA-1-60 with magnetic-activated cell sorting (MACS), and 3 million sorted cells were sub-capsularly transplanted onto kidneys of NSG mice where, after 2 months, they formed a 7 mm × 3 mm × 4 mm myocardial patch resembling the ventricular wall. The graft acquired several features of maturation: expression of ventricular marker (MLC2v), desmosomes, appearance of T-tubule-like structures, and electrophysiological action potential signature consistent with maturation, all this in a non-cardiac environment. We further demonstrated that HVPs transplanted into un-injured hearts of NSG mice remain viable for up to 8 months. Moreover, transplantation of 2 million HVPs largely preserved myocardial contractile function following myocardial infarction. Taken together, our study reaffirms the promising idea of using progenitor cells for regenerative therapy.


Subject(s)
Heart Ventricles/metabolism , Heart Ventricles/physiopathology , LIM-Homeodomain Proteins/metabolism , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Transcription Factors/metabolism , Animals , Cell Differentiation/physiology , Cell Separation/methods , Cells, Cultured , Humans , Male , Mice , Mice, Inbred NOD , Myocardium/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/physiology
6.
Nat Commun ; 9(1): 424, 2018 01 30.
Article in English | MEDLINE | ID: mdl-29382819

ABSTRACT

Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the ß-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and ß-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/ß-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Embryonic Stem Cells/cytology , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Protein p73/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Proteins , Cell Differentiation , DNA-Binding Proteins/metabolism , Embryonic Stem Cells/physiology , Female , Gene Expression Regulation, Developmental , Hippo Signaling Pathway , Humans , Male , Mice, Inbred C57BL , Mice, Inbred CBA , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Phosphoproteins/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction , TEA Domain Transcription Factors , Transcription Factors/metabolism , Tumor Protein p73/genetics , Tumor Suppressor Proteins/genetics , Wnt Proteins/metabolism , YAP-Signaling Proteins , beta Catenin/metabolism
7.
Nat Commun ; 8(1): 921, 2017 10 13.
Article in English | MEDLINE | ID: mdl-29030553

ABSTRACT

Establishment of cell polarity in the mammalian embryo is fundamental for the first cell fate decision that sets aside progenitor cells for both the new organism and the placenta. Yet the sequence of events and molecular mechanism that trigger this process remain unknown. Here, we show that de novo polarisation of the mouse embryo occurs in two distinct phases at the 8-cell stage. In the first phase, an apical actomyosin network is formed. This is a pre-requisite for the second phase, in which the Par complex localises to the apical domain, excluding actomyosin and forming a mature apical cap. Using a variety of approaches, we also show that phospholipase C-mediated PIP2 hydrolysis is necessary and sufficient to trigger the polarisation of actomyosin through the Rho-mediated recruitment of myosin II to the apical cortex. Together, these results reveal the molecular framework that triggers de novo polarisation of the mouse embryo.The molecular trigger that establishes cell polarity in the mammalian embryo is unclear. Here, the authors show that de novo polarisation of the mouse embryo at the 8-cell stage is directed by Phospholipase C and Protein kinase C and occurs in two phases: polarisation of actomyosin followed by the Par complex.


Subject(s)
Actomyosin/metabolism , Cell Polarity , Embryo, Mammalian/metabolism , Protein Kinase C/metabolism , Type C Phospholipases/metabolism , Animals , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Mice, Inbred C57BL , Mice, Inbred CBA , Microscopy, Confocal , Myosin Type II/metabolism , Time-Lapse Imaging/methods
8.
Curr Opin Genet Dev ; 34: 71-6, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26343010

ABSTRACT

Understanding the past is to understand the present. Mammalian life, with all its complexity comes from a humble beginning of a single fertilized egg cell. Achieving this requires an enormous diversification of cellular function, the majority of which is generated through a series of cellular decisions during embryogenesis. The first decisions are made as the embryo prepares for implantation, a process that will require specialization of extra-embryonic lineages while preserving an embryonic one. In this mini-review, we will focus on the mouse as a mammalian model and discuss recent advances in the decision making process of the early embryo.


Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Embryonic Development/genetics , Totipotent Stem Cells/metabolism , Animals , Embryo Implantation/genetics , Embryo, Mammalian , Mice , Signal Transduction/genetics
9.
Nat Protoc ; 9(12): 2732-9, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25356584

ABSTRACT

The implanting mouse blastocyst invades the uterine stroma and undergoes a dramatic transformation into an egg cylinder. The morphogenetic and signaling events during this transition are largely unexplored, as the uterine tissues engulf the embryo. Here we describe a protocol supporting the development of the mouse embryo beyond the blastocyst stage in vitro. We established two types of medium to be applied sequentially, and we used a substrate permitting high-resolution imaging of the transition from blastocyst to egg cylinder. We developed two variants of this protocol: the first starts with intact early blastocysts that upon zona removal can attach to the substrate and develop into egg cylinders after 5 d, and the second starts with late blastocysts that upon dissection of the mural trophectoderm form egg cylinders in only 3 d. This method allows observation of a previously hidden period of development, and it provides a platform for novel research into peri-implantation embryogenesis and beyond.


Subject(s)
Blastocyst/cytology , Embryo Culture Techniques/methods , Animals , Blastocyst/physiology , Cell Adhesion , Ectoderm/cytology , Embryo Culture Techniques/instrumentation , Embryo Implantation , Female , Mice, Inbred C57BL
10.
Philos Trans R Soc Lond B Biol Sci ; 369(1657)2014 Dec 05.
Article in English | MEDLINE | ID: mdl-25349447

ABSTRACT

A critical point in mammalian development is when the early embryo implants into its mother's uterus. This event has historically been difficult to study due to the fact that it occurs within the maternal tissue and therefore is hidden from view. In this review, we discuss how the mouse embryo is prepared for implantation and the molecular mechanisms involved in directing and coordinating this crucial event. Prior to implantation, the cells of the embryo are specified as precursors of future embryonic and extra-embryonic lineages. These preimplantation cell fate decisions rely on a combination of factors including cell polarity, position and cell-cell signalling and are influenced by the heterogeneity between early embryo cells. At the point of implantation, signalling events between the embryo and mother, and between the embryonic and extraembryonic compartments of the embryo itself, orchestrate a total reorganization of the embryo, coupled with a burst of cell proliferation. New developments in embryo culture and imaging techniques have recently revealed the growth and morphogenesis of the embryo at the time of implantation, leading to a new model for the blastocyst to egg cylinder transition. In this model, pluripotent cells that will give rise to the fetus self-organize into a polarized three-dimensional rosette-like structure that initiates egg cylinder formation.


Subject(s)
Body Patterning/physiology , Cell Differentiation/physiology , Cell Lineage/physiology , Embryo Implantation/physiology , Embryo, Mammalian/embryology , Morphogenesis/physiology , Animals , Mice , Models, Biological
11.
Nat Commun ; 4: 2251, 2013.
Article in English | MEDLINE | ID: mdl-23903990

ABSTRACT

Cell identity is specified in the early mammalian embryo by the generation of precursors for two cell lineages: the pluripotent inner cell mass and differentiating trophectoderm. Here we identify Angiomotin as a key regulator of this process. We show that the loss of Angiomotin, together with Angiomotin-like 2, leads to differentiation of inner cell mass cells and compromised peri-implantation development. We show that Angiomotin regulates localization of Yap, and Yap-binding motifs are required for full activity of Angiomotin. Importantly, we also show that Angiomotin function can compensate for the absence of Lats1/2 kinases, indicating the ability of Angiomotin to bypass the classical Hippo pathway for Yap regulation. In polarized outside cells, Angiomotin localizes apically, pointing to the importance of cell polarity in regulating Yap to promote differentiation. We propose that both Hippo pathway-dependent and Hippo pathway-independent mechanisms regulate Yap localization to set apart pluripotent and differentiated lineages in the pre-implantation mouse embryo.


Subject(s)
Cell Differentiation , Cell Lineage , Embryo, Mammalian/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Microfilament Proteins/metabolism , Pluripotent Stem Cells/cytology , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Amino Acid Motifs , Angiomotins , Animals , Blastocyst/cytology , Blastocyst/metabolism , Ectoderm/cytology , Ectoderm/metabolism , Humans , Intercellular Signaling Peptides and Proteins/deficiency , Mice , Microfilament Proteins/deficiency , Models, Biological , Pluripotent Stem Cells/metabolism , Protein Isoforms/metabolism
12.
Genes Dev ; 25(19): 2031-40, 2011 Oct 01.
Article in English | MEDLINE | ID: mdl-21979916

ABSTRACT

DNA-dependent protein kinase (DNA-PK) is a central regulator of DNA double-strand break (DSB) repair; however, the identity of relevant DNA-PK substrates has remained elusive. NR4A nuclear orphan receptors function as sequence-specific DNA-binding transcription factors that participate in adaptive and stress-related cell responses. We show here that NR4A proteins interact with the DNA-PK catalytic subunit and, upon exposure to DNA damage, translocate to DSB foci by a mechanism requiring the activity of poly(ADP-ribose) polymerase-1 (PARP-1). At DNA repair foci, NR4A is phosphorylated by DNA-PK and promotes DSB repair. Notably, NR4A transcriptional activity is entirely dispensable in this function, and core components of the DNA repair machinery are not transcriptionally regulated by NR4A. Instead, NR4A functions directly at DNA repair sites by a process that requires phosphorylation by DNA-PK. Furthermore, a severe combined immunodeficiency (SCID)-causing mutation in the human gene encoding the DNA-PK catalytic subunit impairs the interaction and phosphorylation of NR4A at DSBs. Thus, NR4As represent an entirely novel component of DNA damage response and are substrates of DNA-PK in the process of DSB repair.


Subject(s)
Calcium-Binding Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Repair , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Animals , Cell Line , Cells, Cultured , Gene Knockout Techniques , Humans , Mice , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Phosphorylation , Protein Transport , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/physiopathology
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