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Angelova DM, Tsolova A, Prater M, Ballasy N, Bacon W, Hamilton RS, Blackwell D, Yu Z, Li X, Liu X, Hemberger M, Charnock-Jones DS. Single-cell RNA sequencing identifies CXADR as a fate determinant of the placental exchange surface. Nat Commun 2025; 16:142. [PMID: 39747179 PMCID: PMC11695997 DOI: 10.1038/s41467-024-55597-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Accepted: 12/16/2024] [Indexed: 01/04/2025] Open
Abstract
The placenta is the critical interface between mother and fetus, and consequently, placental dysfunction underlies many pregnancy complications. Placental formation requires an adequate expansion of trophoblast stem and progenitor cells followed by finely tuned lineage specification events. Here, using single-cell RNA sequencing of mouse trophoblast stem cells during the earliest phases of differentiation, we identify gatekeepers of the stem cell state, notably Nicol1, and uncover unsuspected trajectories of cell lineage diversification as well as regulators of lineage entry points. We show that junctional zone precursors and precursors of one of the two syncytial layers of the mouse placental labyrinth, the Syncytiotrophoblast-I lineage, initially share similar trajectories. Importantly, our functional analysis of one such lineage precursor marker, CXADR, demonstrates that this cell surface protein regulates the differentiation dynamics between the two syncytial layers of the mouse labyrinth, ensuring the correct establishment of the placental exchange surface. Deciphering the mechanisms underlying trophoblast lineage specification will inform our understanding of human pregnancy in health and disease.
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Affiliation(s)
- Dafina M Angelova
- Department of Obstetrics and Gynaecology, University of Cambridge, NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom
- Loke Centre for Trophoblast Research, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom
| | - Aleksandra Tsolova
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
- Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
| | - Malwina Prater
- Loke Centre for Trophoblast Research, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom
- Functional Genomics Centre, Cancer Research Horizons, Milner Therapeutics Institute, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge, United Kingdom
| | - Noura Ballasy
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
- Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
| | - Wendi Bacon
- Loke Centre for Trophoblast Research, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom
- School of Life, Health & Chemical Sciences, The Open University, Milton Keynes, United Kingdom
| | - Russell S Hamilton
- Loke Centre for Trophoblast Research, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom
| | - Danielle Blackwell
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
- Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada
| | - Ziyi Yu
- College of Chemical Engineering, Nanjing Tech University, Nanjing, People's Republic of China
| | - Xin Li
- Sphere Fluidics Ltd., Building One, Granta Centre, Granta Park, Great Abington, Cambridge, England, United Kingdom
| | - Xin Liu
- Sphere Fluidics Ltd., Building One, Granta Centre, Granta Park, Great Abington, Cambridge, England, United Kingdom
| | - Myriam Hemberger
- Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.
- Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.
| | - D Stephen Charnock-Jones
- Department of Obstetrics and Gynaecology, University of Cambridge, NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.
- Loke Centre for Trophoblast Research, Department of Physiology, Development, and Neuroscience, University of Cambridge, Cambridge, United Kingdom.
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Ballasy N, Apantaku I, Dean W, Hemberger M. Off to a good start: The importance of the placental exchange surface - Lessons from the mouse. Dev Biol 2025; 517:248-264. [PMID: 39491740 DOI: 10.1016/j.ydbio.2024.10.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2024] [Revised: 10/04/2024] [Accepted: 10/31/2024] [Indexed: 11/05/2024]
Abstract
The role of the chorio-allantoic placenta as the critical nutrient- and oxygen-supplying organ to nourish the demands of the fetus has been well recognized. This function relies on the successful establishment of the placental feto-maternal exchange unit, or interhaemal barrier, across which all nutrients as well as waste products must pass to cross from the maternal to the fetal blood circulation, or vice versa, respectively. As a consequence, defects in the establishment of this elaborate interface lead to fetal growth retardation or even embryonic lethality, depending on the severity of the defect. Beyond this essential role, however, it has also emerged that the functionality of the feto-maternal interface dictates the proper development of specific embryonic organs, with tightest links observed to the formation of the heart. In this article, we build on the foundational strength of the mouse as experimental model in which the placental causality of embryonic defects can be genetically proven. We discuss in detail the formation of the interhaemal barrier that makes up the labyrinth layer of the murine placenta, including insights into drivers of its formation and the interdependence of the cell types that make up this essential interface, from in vivo and in vitro data using mouse trophoblast stem cells. We highlight mouse genetic tools that enable the elucidation of cause-effect relationships between defects driven by either the trophoblast cells of the placenta or by embryonic cell types. We specifically emphasize gene knockouts for which a placental causality of embryonic heart defects has been demonstrated. This in-depth perspective provides much-needed insights while highlighting remaining gaps in knowledge that are essential for gaining a better understanding of the multi-facetted roles of the placenta in setting us up for a healthy start in life well beyond nutritional support alone.
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Affiliation(s)
- Noura Ballasy
- Dept. of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada; Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada
| | - Ifeoluwa Apantaku
- Dept. of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada; Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada
| | - Wendy Dean
- Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada; Dept. of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada
| | - Myriam Hemberger
- Dept. of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada; Alberta Children's Hospital Research Institute, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada.
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Waheed‐Ullah Q, Wilsdon A, Abbad A, Rochette S, Bu'Lock F, Hitz M, Dombrowsky G, Cuello F, Brook JD, Loughna S. Effect of deletion of the protein kinase PRKD1 on development of the mouse embryonic heart. J Anat 2024; 245:70-83. [PMID: 38419169 PMCID: PMC11161829 DOI: 10.1111/joa.14033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2023] [Revised: 02/14/2024] [Accepted: 02/15/2024] [Indexed: 03/02/2024] Open
Abstract
Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes-CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1) carrying deletion of exon 2, causing loss of function. High-resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT-qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT-qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD.
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Affiliation(s)
- Qazi Waheed‐Ullah
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
| | - Anna Wilsdon
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
| | - Aseel Abbad
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
| | - Sophie Rochette
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
| | - Frances Bu'Lock
- East Midlands Congenital Heart CentreUniversity Hospitals of Leicester NHS TrustLeicesterUK
| | - Marc‐Phillip Hitz
- Institute of Medical GeneticsCarl von Ossietzky University OldenburgOldenburgGermany
| | - Gregor Dombrowsky
- Institute of Medical GeneticsCarl von Ossietzky University OldenburgOldenburgGermany
| | - Friederike Cuello
- Institute of Experimental Pharmacology and Toxicology, Cardiovascular Research CenterUniversity Medical Center Hamburg‐EppendorfHamburgGermany
- DZHK (German Center for Cardiovascular Research), partner site Hamburg/Kiel/LübeckUniversity Medical Center Hamburg‐EppendorfHamburgGermany
| | - J. David Brook
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
| | - Siobhan Loughna
- School of Life Sciences, Faculty of Medicine and Health SciencesUniversity of NottinghamNottinghamUK
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Yang D, Jian Z, Tang C, Chen Z, Zhou Z, Zheng L, Peng X. Zebrafish Congenital Heart Disease Models: Opportunities and Challenges. Int J Mol Sci 2024; 25:5943. [PMID: 38892128 PMCID: PMC11172925 DOI: 10.3390/ijms25115943] [Citation(s) in RCA: 7] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2024] [Revised: 05/18/2024] [Accepted: 05/23/2024] [Indexed: 06/21/2024] Open
Abstract
Congenital heart defects (CHDs) are common human birth defects. Genetic mutations potentially cause the exhibition of various pathological phenotypes associated with CHDs, occurring alone or as part of certain syndromes. Zebrafish, a model organism with a strong molecular conservation similar to humans, is commonly used in studies on cardiovascular diseases owing to its advantageous features, such as a similarity to human electrophysiology, transparent embryos and larvae for observation, and suitability for forward and reverse genetics technology, to create various economical and easily controlled zebrafish CHD models. In this review, we outline the pros and cons of zebrafish CHD models created by genetic mutations associated with single defects and syndromes and the underlying pathogenic mechanism of CHDs discovered in these models. The challenges of zebrafish CHD models generated through gene editing are also discussed, since the cardiac phenotypes resulting from a single-candidate pathological gene mutation in zebrafish might not mirror the corresponding human phenotypes. The comprehensive review of these zebrafish CHD models will facilitate the understanding of the pathogenic mechanisms of CHDs and offer new opportunities for their treatments and intervention strategies.
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Michalski MN, Williams BO. The Past, Present, and Future of Genetically Engineered Mouse Models for Skeletal Biology. Biomolecules 2023; 13:1311. [PMID: 37759711 PMCID: PMC10526739 DOI: 10.3390/biom13091311] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2023] [Revised: 08/25/2023] [Accepted: 08/25/2023] [Indexed: 09/29/2023] Open
Abstract
The ability to create genetically engineered mouse models (GEMMs) has exponentially increased our understanding of many areas of biology. Musculoskeletal biology is no exception. In this review, we will first discuss the historical development of GEMMs and how these developments have influenced musculoskeletal disease research. This review will also update our 2008 review that appeared in BONEKey, a journal that is no longer readily available online. We will first review the historical development of GEMMs in general, followed by a particular emphasis on the ability to perform tissue-specific (conditional) knockouts focusing on musculoskeletal tissues. We will then discuss how the development of CRISPR/Cas-based technologies during the last decade has revolutionized the generation of GEMMs.
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Affiliation(s)
- Megan N. Michalski
- Department of Cell Biology, Van Andel Institute, Grand Rapids, MI 49503, USA;
| | - Bart O. Williams
- Department of Cell Biology, Van Andel Institute, Grand Rapids, MI 49503, USA;
- Core Technologies and Services, Van Andel Institute, Grand Rapids, MI 49503, USA
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Loss of GLTSCR1 causes congenital heart defects by regulating NPPA transcription. Angiogenesis 2023; 26:217-232. [PMID: 36745292 PMCID: PMC10119265 DOI: 10.1007/s10456-023-09869-6] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Accepted: 01/23/2023] [Indexed: 02/07/2023]
Abstract
Precise and specific spatiotemporal domains of gene expression regulation are critical for embryonic development. Recent studies have identified GLTSCR1 as a gene transcriptional elongation regulator in cancer research. However, the function of GLTSCR1, especially in embryonic development, remains poorly understood. Here, we found that GLTSCR1 was essential for cardiac development because Gltscr1 knockout (Gltscr1-/-) led to embryonic lethality in mice with severe congenital heart defects (CHDs). Ventricular septal defect and double outflow right ventricular were also observed in neural crest cells with conditional deletion of Gltscr1, which were associated with neonatal lethality in mice. Mechanistically, GLTSCR1 deletion promoted NPPA expression by coordinating the CHD risk G allele of rs56153133 in the NPPA enhancer and releasing the transcription factor ZNF740-binding site on the NPPA promoter. These findings demonstrated that GLTSCR1 acts as a candidate CHD-related gene.
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Downs KM. The mouse allantois: new insights at the embryonic-extraembryonic interface. Philos Trans R Soc Lond B Biol Sci 2022; 377:20210251. [PMID: 36252214 PMCID: PMC9574631 DOI: 10.1098/rstb.2021.0251] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Accepted: 01/20/2022] [Indexed: 12/23/2022] Open
Abstract
During the early development of Placentalia, a distinctive projection emerges at the posterior embryonic-extraembryonic interface of the conceptus; its fingerlike shape presages maturation into the placental umbilical cord, whose major role is to shuttle fetal blood to and from the chorion for exchange with the mother during pregnancy. Until recently, the biology of the cord's vital vascular anlage, called the body stalk/allantois in humans and simply the allantois in rodents, has been largely unknown. Here, new insights into the development of the mouse allantois are featured, from its origin and mechanism of arterial patterning through its union with the chorion. Key to generating the allantois and its critical functions are the primitive streak and visceral endoderm, which together are sufficient to create the entire fetal-placental connection. Their newly discovered roles at the embryonic-extraembryonic interface challenge conventional wisdom, including the physical limits of the primitive streak, its function as sole purveyor of mesoderm in the mouse, potency of visceral endoderm, and the putative role of the allantois in the germ line. With this working model of allantois development, understanding a plethora of hitherto poorly understood orphan diseases in humans is now within reach. This article is part of the theme issue 'Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom'.
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Affiliation(s)
- Karen M. Downs
- Department of Cell and Regenerative Biology, University of Wisconsin-Madison School of Medicine and Public Health, 1111 Highland Avenue, Madison, WI 53705, USA
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Puscheck EE, Ruden X, Singh A, Abdulhasan M, Ruden DM, Awonuga AO, Rappolee DA. Using high throughput screens to predict miscarriages with placental stem cells and long-term stress effects with embryonic stem cells. Birth Defects Res 2022; 114:1014-1036. [PMID: 35979652 PMCID: PMC10108263 DOI: 10.1002/bdr2.2079] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 07/25/2022] [Accepted: 07/26/2022] [Indexed: 11/10/2022]
Abstract
A problem in developmental toxicology is the massive loss of life from fertilization through gastrulation, and the surprising lack of knowledge of causes of miscarriage. Half to two-thirds of embryos are lost, and environmental and genetic causes are nearly equal. Simply put, it can be inferred that this is a difficult period for normal embryos, but that environmental stresses may cause homeostatic responses that move from adaptive to maladaptive with increasing exposures. At the lower 50% estimate, miscarriage causes greater loss-of-life than all cancers combined or of all cardio- and cerebral-vascular accidents combined. Surprisingly, we do not know if miscarriage rates are increasing or decreasing. Overshadowed by the magnitude of miscarriages, are insufficient data on teratogenic or epigenetic imbalances in surviving embryos and their stem cells. Superimposed on the difficult normal trajectory for peri-gastrulation embryos are added malnutrition, hormonal, and environmental stresses. An overarching hypothesis is that high throughput screens (HTS) using cultured viable reporter embryonic and placental stem cells (e.g., embryonic stem cells [ESC] and trophoblast stem cells [TSC] that report status using fluorescent reporters in living cells) from the pre-gastrulation embryo will most rapidly test a range of hormonal, environmental, nutritional, drug, and diet supplement stresses that decrease stem cell proliferation and imbalance stemness/differentiation. A second hypothesis is that TSC respond with greater sensitivity in magnitude to stress that would cause miscarriage, but ESC are stress-resistant to irreversible stemness loss and are best used to predict long-term health defects. DevTox testing needs more ESC and TSC HTS to model environmental stresses leading to miscarriage or teratogenesis and more research on epidemiology of stress and miscarriage. This endeavor also requires a shift in emphasis on pre- and early gastrulation events during the difficult period of maximum loss by miscarriage.
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Affiliation(s)
- Elizabeth E Puscheck
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
- Reproductive Stress 3M Inc, Grosse Pointe Farms, Michigan, USA
- Invia Fertility Clinics, Hoffman Estates, Illinois, USA
| | - Ximena Ruden
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
| | - Aditi Singh
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
- Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, USA
| | - Mohammed Abdulhasan
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
- Reproductive Stress 3M Inc, Grosse Pointe Farms, Michigan, USA
| | - Douglas M Ruden
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
- Invia Fertility Clinics, Hoffman Estates, Illinois, USA
- Institute for Environmental Health Science, Wayne State University School of Medicine, Detroit, Michigan, USA
| | - Awoniyi O Awonuga
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
| | - Daniel A Rappolee
- CS Mott Center for Human Growth and Development, Department of Ob/Gyn, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan, USA
- Reproductive Stress 3M Inc, Grosse Pointe Farms, Michigan, USA
- Invia Fertility Clinics, Hoffman Estates, Illinois, USA
- Institute for Environmental Health Science, Wayne State University School of Medicine, Detroit, Michigan, USA
- Program for Reproductive Sciences and Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, USA
- Department of Biology, University of Windsor, Windsor, Ontario, Canada
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Zhu Q, Zhou Y, Ding J, Chen L, Liu J, Zhou T, Bian W, Ding G, Li G. Screening of Candidate Pathogenic Genes for Spontaneous Abortion using Whole Exome Sequencing. Comb Chem High Throughput Screen 2021; 25:1462-1473. [PMID: 34225611 DOI: 10.2174/1386207324666210628115715] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 04/19/2021] [Accepted: 05/10/2021] [Indexed: 11/22/2022]
Abstract
BACKGROUND Spontaneous abortion is a common disease in obstetrics and reproduction. OBJECTIVE This study aimed to screen candidate pathogenic genes for spontaneous abortion using whole-exome sequencing. METHODS Genomic DNA was extracted from abortion tissues of spontaneous abortion patients and sequenced using the Illumina HiSeq2500 high-throughput sequencing platform. Whole exome sequencing was performed to select harmful mutations, including SNP and insertion and deletion sites, associated with spontaneous abortion. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses and gene fusion analyses were performed. MUC3A and PDE4DIP were two novel mutation genes that were screened and verified by PCR in abortion tissues of patients. RESULTS A total of 83,633 SNPs and 13,635 Indel mutations were detected, of which 29172 SNPs and 3093 Indels were screened as harmful mutations. The 7 GO-BP, 4 GO-CC, 9 GO-MF progress, and 3 KEGG pathways were enriched in GO and KEGG pathway analyses. A total of 746 gene fusion mutations were obtained, involving 492 genes. MUC3A and PDE4DIP were used for PCR verification because of their high number of mutation sites in all samples. CONCLUSION There are extensive SNPs and Indel mutations in the genome of spontaneous abortion tissues, and the effect of these gene mutations on spontaneous abortion needs further experimental verification.
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Affiliation(s)
- Qingwen Zhu
- Nantong Municipal Maternal and Child Health Hospital, Nantong, 226010, China
| | - Yiwen Zhou
- Shanghai Biological Information Research Center, Zhangjiang Hi-tech Park, Shanghai, 201203, China
| | - Jiayi Ding
- Reproductive Medicine Center, Nantong Municipal Maternal and Child Health Hospital, Nantong, 226010, China
| | - Li Chen
- Reproductive Medicine Center, Nantong Municipal Maternal and Child Health Hospital, Nantong, 226010, China
| | - Jia Liu
- Shanghai Biological Information Research Center, Zhangjiang Hi-tech Park, Shanghai, 201203, China
| | - Tao Zhou
- Reproductive Medicine Center, Nantong Municipal Maternal and Child Health Hospital, Nantong, 226010, China
| | - Wenjun Bian
- Prenatal Screening and Diagnosis Center, Nantong Municipal Maternal and Child Health Hospital, Nantong, 226010, China
| | - Guohui Ding
- Shanghai Biological Information Research Center, Zhangjiang Hi-tech Park, Shanghai, 201203, China
| | - Guang Li
- Shanghai Biological Information Research Center, Zhangjiang Hi-tech Park, Shanghai, 201203, China
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Díaz-Hernández I, Alecsandru D, García-Velasco JA, Domínguez F. Uterine natural killer cells: from foe to friend in reproduction. Hum Reprod Update 2021; 27:720-746. [PMID: 33528013 DOI: 10.1093/humupd/dmaa062] [Citation(s) in RCA: 65] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Revised: 12/15/2020] [Indexed: 12/21/2022] Open
Abstract
BACKGROUND Recurrent miscarriage and pre-eclampsia are common reproductive disorders, but their causes are often unknown. Recent evidence has provided new insight into immune system influences in reproductive disorders. A subset of lymphocytes of the innate immune system known as uterine natural killer (uNK) cells are now recognized as fundamental to achieving embryo implantation and successful pregnancy, but were initially attributed a bad reputation. Indeed, immune therapies have been developed to treat the 'exaggerated' immune response from uNK cells. These treatments have been based on studies of peripheral blood natural killer (pbNK) cells. However, uNK cells and pbNK cells have different phenotypic and functional characteristics. The functions of uNK cells are closely related to their interactions with the extravillous trophoblast cells (EVTs) and spiral arteries, which underlie an essential role in regulating vascular function, controlling trophoblast invasion and promoting placental development. EVTs express MHC molecules of class I HLA-C/E/G/F, while uNK cells express, among other receptors, killer cell immunoglobulin-like receptors (KIRs) that bind to HLA-C or CD94/NKG2A inhibitory receptors, and then bind HLA-E. Associations of certain KIR/HLA-C combinations with recurrent miscarriage, pre-eclampsia, and foetal growth restriction and the interactions between uNK cells, trophoblasts and vascular cells have led to the hypothesis that uNK cells may play a role in embryo implantation. OBJECTIVE AND RATIONALE Our objective was to review the evolution of our understanding of uNK cells, their functions, and their increasingly relevant role in reproduction. SEARCH METHODS Relevant literature through June 2020 was retrieved using Google Scholar and PubMed. Search terms comprised uNK cells, human pregnancy, reproductive failure, maternal KIR and HLA-C, HLA-E/G/F in EVT cells, angiogenic cytokines, CD56+ NK cells, spiral artery, oestrogen and progesterone receptors, KIR haplotype and paternal HLA-C2. OUTCOMES This review provides key insights into the evolving conceptualization of uNK cells, from their not-so-promising beginnings to now, when they are considered allies in reproduction. We synthesized current knowledge about uNK cells, their involvement in reproduction and their main functions in placental vascular remodeling and trophoblast invasion. One of the issues that this review presents is the enormous complexity involved in studying the immune system in reproduction. The complexity in the immunology of the maternal-foetal interface lies in the great variety of participating molecules, the processes and interactions that occur at different levels (molecular, cellular, tissue, etc.) and the great diversity of genetic combinations that are translated into different types of responses. WIDER IMPLICATIONS Insights into uNK cells could offer an important breakthrough for ART outcomes, since each patient could be assessed based on the combination of HLA and its receptors in their uNK cells, evaluating the critical interactions at the materno-foetal interface. However, owing to the technical challenges in studying uNK cells in vivo, there is still much knowledge to gain, particularly regarding their exact origin and functions. New studies using novel molecular and genetic approaches can facilitate the identification of mechanisms by which uNK cells interact with other cells at the materno-foetal interface, perhaps translating this knowledge into clinical applicability.
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Affiliation(s)
| | - Diana Alecsandru
- Department of Immunology and Department of Reproductive Endocrinology and Infertility, Instituto Valenciano de Infertilidad-Madrid, Rey Juan Carlos University (IVI), Madrid 28023, Spain
| | - Juan Antonio García-Velasco
- Department of Immunology and Department of Reproductive Endocrinology and Infertility, Instituto Valenciano de Infertilidad-Madrid, Rey Juan Carlos University (IVI), Madrid 28023, Spain
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Auclair N, Sané AT, Ahmarani L, Patey N, Beaulieu JF, Peretti N, Spahis S, Levy E. Sar1b mutant mice recapitulate gastrointestinal abnormalities associated with chylomicron retention disease. J Lipid Res 2021; 62:100085. [PMID: 33964306 PMCID: PMC8175419 DOI: 10.1016/j.jlr.2021.100085] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 04/15/2021] [Accepted: 04/17/2021] [Indexed: 11/17/2022] Open
Abstract
Chylomicron retention disease (CRD) is an autosomal recessive disorder associated with biallelic Sar1b mutations leading to defects in intracellular chylomicron (CM) trafficking and secretion. To date, a direct cause-effect relationship between CRD and Sar1b mutation has not been established, but genetically modified animal models provide an opportunity to elucidate unrecognized aspects of these mutations. To examine the physiological role and molecular mechanisms of Sar1b function, we generated mice expressing either a targeted deletion or mutation of human Sar1b using the CRISPR-Cas9 system. We found that deletion or mutation of Sar1b in mice resulted in late-gestation lethality of homozygous embryos. Moreover, compared with WT mice, heterozygotes carrying a single disrupted Sar1b allele displayed lower plasma levels of triglycerides, total cholesterol, and HDL-cholesterol, along with reduced CM secretion following gastric lipid gavage. Similarly, decreased expression of apolipoprotein B and microsomal triglyceride transfer protein was observed in correlation with the accumulation of mucosal lipids. Inefficient fat absorption in heterozygotes was confirmed via an increase in fecal lipid excretion. Furthermore, genetically modified Sar1b affected intestinal lipid homeostasis as demonstrated by enhanced fatty acid β-oxidation and diminished lipogenesis through the modulation of transcription factors. This is the first reported mammalian animal model with human Sar1b genetic defects, which reproduces some of the characteristic CRD features and provides a direct cause-effect demonstration.
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Affiliation(s)
- Nickolas Auclair
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada; Department of Pharmacology & Physiology, Université de Montréal, Montreal, Quebec, Canada
| | - Alain T Sané
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada
| | - Lena Ahmarani
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada; Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada
| | - Nathalie Patey
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada; Department of Pathology, Université de Montréal, Montreal, Quebec, Canada
| | - Jean-François Beaulieu
- Laboratory of Intestinal Physiopathology, Department of Immunology and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada
| | - Noel Peretti
- Department of Pediatric Gastroenterology-Hepatology and Nutrition, Laboratory INSERM 1060 Cardiovascular Metabolism Endocrinology and Nutrition CarMEN, Lyon, France
| | - Schohraya Spahis
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada; Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada
| | - Emile Levy
- Research Center, CHU Ste-Justine, Université de Montréal, Montreal, Quebec, Canada; Department of Pharmacology & Physiology, Université de Montréal, Montreal, Quebec, Canada; Department of Nutrition, Université de Montréal, Montreal, Quebec, Canada.
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12
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setd2 knockout zebrafish is viable and fertile: differential and developmental stress-related requirements for Setd2 and histone H3K36 trimethylation in different vertebrate animals. Cell Discov 2020; 6:72. [PMID: 33088589 PMCID: PMC7573620 DOI: 10.1038/s41421-020-00203-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Accepted: 08/01/2020] [Indexed: 12/21/2022] Open
Abstract
Setd2 is the only enzyme that catalyzes histone H3 lysine 36 trimethylation (H3K36me3) on virtually all actively transcribed protein-coding genes, and this mechanism is evolutionarily conserved from yeast to human. Despite this widespread and conserved activity, Setd2 and H3K36me3 are dispensable for normal growth of yeast but are absolutely required for mammalian embryogenesis, such as oocyte maturation and embryonic vasculogenesis in mice, raising a question of how the functional requirements of Setd2 in specific developmental stages have emerged through evolution. Here, we explored this issue by studying the essentiality and function of Setd2 in zebrafish. Surprisingly, the setd2-null zebrafish are viable and fertile. They show Mendelian birth ratio and normal embryogenesis without vascular defect as seen in mice; however, they have a small body size phenotype attributed to insufficient energy metabolism and protein synthesis, which is reversable in a nutrition-dependent manner. Unlike the sterile Setd2-null mice, the setd2-null zebrafish can produce functional sperms and oocytes. Nonetheless, related to the requirement of maternal Setd2 for oocyte maturation in mice, the second generation of setd2-null zebrafish that carry no maternal setd2 show decreased survival rate and a developmental delay at maternal-to-zygotic transition. Taken together, these results indicate that, while the phenotypes of the setd2-null zebrafish and mice are apparently different, they are matched in parallel as the underlying mechanisms are evolutionarily conserved. Thus, the differential requirements of Setd2 may reflect distinct viability thresholds that associate with intrinsic and/or extrinsic stresses experienced by the organism through development, and these epigenetic regulatory mechanisms may serve as a reserved source supporting the evolution of life from simplicity to complexity.
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13
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Chin HJ, Lee SY, Lee D. Tamoxifen-inducible cardiac-specific Cre transgenic mouse using VIPR2 intron. Lab Anim Res 2020; 36:31. [PMID: 32983955 PMCID: PMC7493340 DOI: 10.1186/s42826-020-00065-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2020] [Accepted: 09/07/2020] [Indexed: 11/20/2022] Open
Abstract
Genetically engineered mouse models through gene deletion are useful tools for analyzing gene function. To delete a gene in a certain tissue temporally, tissue-specific and tamoxifen-inducible Cre transgenic mice are generally used. Here, we generated transgenic mouse with cardiac-specific expression of Cre recombinase fused to a mutant estrogen ligand-binding domain (ERT2) on both N-terminal and C-terminal under the regulatory region of human vasoactive intestinal peptide receptor 2 (VIPR2) intron and Hsp68 promoter (VIPR2-ERT2CreERT2). In VIPR2-ERT2CreERT2 transgenic mice, mRNA for Cre gene was highly expressed in the heart. To further reveal heart-specific Cre expression, VIPR2-ERT2CreERT2 mice mated with ROSA26-lacZ reporter mice were examined by X-gal staining. Results of X-gal staining revealed that Cre-dependent recombination occurred only in the heart after treatment with tamoxifen. Taken together, these results demonstrate that VIPR2-ERT2CreERT2 transgenic mouse is a useful model to unveil a specific gene function in the heart.
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Affiliation(s)
- Hyun Jung Chin
- Department of Life Science, Ewha Womans University, Ewhayeodae-gil 52, Seodaemun-gu, Seoul, 03760 South Korea
| | - So-Young Lee
- Department of Life Science, Ewha Womans University, Ewhayeodae-gil 52, Seodaemun-gu, Seoul, 03760 South Korea
| | - Daekee Lee
- Department of Life Science, Ewha Womans University, Ewhayeodae-gil 52, Seodaemun-gu, Seoul, 03760 South Korea
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14
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Shibata S, Kobayashi EH, Kobayashi N, Oike A, Okae H, Arima T. Unique features and emerging in vitro models of human placental development. Reprod Med Biol 2020; 19:301-313. [PMID: 33071632 PMCID: PMC7542016 DOI: 10.1002/rmb2.12347] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 08/21/2020] [Accepted: 08/21/2020] [Indexed: 12/13/2022] Open
Abstract
Background The placenta is an essential organ for the normal development of mammalian fetuses. Most of our knowledge on the molecular mechanisms of placental development has come from the analyses of mice, especially histopathological examination of knockout mice. Choriocarcinoma and immortalized cell lines have also been used for basic research on the human placenta. However, these cells are quite different from normal trophoblast cells. Methods In this review, we first provide an overview of mouse and human placental development with particular focus on the differences in the anatomy, transcription factor networks, and epigenetic characteristics between these species. Next, we discuss pregnancy complications associated with abnormal placentation. Finally, we introduce emerging in vitro models to study the human placenta, including human trophoblast stem (TS) cells, trophoblast and endometrium organoids, and artificial embryos. Main findings The placental structure and development differ greatly between humans and mice. The recent establishment of human TS cells and trophoblast and endometrial organoids enhances our understanding of the mechanisms underlying human placental development. Conclusion These in vitro models will greatly advance our understanding of human placental development and potentially contribute to the elucidation of the causes of infertility and other pregnancy complications.
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Affiliation(s)
- Shun Shibata
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
| | - Eri H Kobayashi
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
| | - Norio Kobayashi
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
| | - Akira Oike
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
| | - Hiroaki Okae
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
| | - Takahiro Arima
- Department of Informative Genetics Tohoku University Graduate School of Medicine Sendai Japan
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15
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Tyser RCV, Srinivas S. The First Heartbeat-Origin of Cardiac Contractile Activity. Cold Spring Harb Perspect Biol 2020; 12:cshperspect.a037135. [PMID: 31767652 DOI: 10.1101/cshperspect.a037135] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The amniote embryonic heart starts as a crescent of mesoderm that transitions through a midline linear heart tube in the course of developing into the four chambered heart. It is unusual in having to contract rhythmically while still undergoing extensive morphogenetic remodeling. Advances in imaging have allowed us to determine when during development this contractile activity starts. In the mouse, focal regions of contractions can be detected as early as the cardiac crescent stage. Calcium transients, required to trigger contraction, can be detected even earlier, prior to contraction. In this review, we outline what is currently known about how this early contractile function is initiated and the impact early contractile function has on cardiac development.
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Affiliation(s)
- Richard C V Tyser
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
| | - Shankar Srinivas
- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom
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16
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Zhang S, Song Z, An L, Liu X, Hu XW, Naz A, Zhou R, Guo X, He L, Zhu H. WD40 repeat and FYVE domain containing 3 is essential for cardiac development. Cardiovasc Res 2020; 115:1320-1331. [PMID: 30428088 DOI: 10.1093/cvr/cvy285] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/30/2018] [Revised: 10/22/2018] [Accepted: 11/13/2018] [Indexed: 01/02/2023] Open
Abstract
AIMS WD40 repeat and FYVE domain containing 3 (WDFY3) is an adaptor protein involved in selective degradation of protein aggregates by autophagy. Recent studies have revealed that Wdfy3 is critical in the regulation of brain development and osteoclastogenesis in vivo. However, the function of Wdfy3 in cardiac development remains completely unknown. In this study, we explore the role of Wdfy3 in cardiac morphogenesis using Wdfy3-deficient mice. METHODS AND RESULTS Wdfy3 was expressed in the developing heart in mice and peaked at embryonic day 12.5 (E12.5). Loss of Wdfy3 in mice led to embryonic and neonatal lethality. Wdfy3-deficient mice displayed various congenital heart defects including membranous ventricular septal defect (VSD), aortic overriding (AO), double outlet right ventricle (DORV), thinning of ventricular wall, ventricular dilation, and disorganized ventricular trabeculation at E14.5. Cell proliferation was reduced in the hearts from Wdfy3-deficient mice at E12.5 and E14.5, which was associated with enhanced p21 expression. Cardiomyocyte differentiation was diminished as demonstrated by reduced Myh6 and MLC2v in Wdfy3-deficient mice at E14.5. In addition, Nkx2-5 and Mef2c, two cardiac transcription factors regulating cardiomyocyte differentiation, were decreased in Wdfy3-deficient mice at E14.5. Apoptotic cell death remained unaltered. These data suggest that reduced cell proliferation and cardiomyocyte differentiation contribute to cardiac defects in Wdfy3-deficient mice. Mechanistically, loss of Wdfy3 led to a reduction in protein levels of Notch 1 intracellular domain and its downstream targets Hes1 and Hey1, which was accompanied with enhanced full-length Notch1 protein levels. In vitro luciferase assay showed that Wdfy3 deficiency induced activity of p21 promoter, while diminished activity of Hes1 promoter through modulation of Notch1 signalling. Moreover, Wdfy3 was co-localized with Notch1 in primary embryonic cardiomyocytes. Endogenous Wdfy3 physically interacted with full-length Notch1 in the developing heart. These results suggest that Notch1 signalling is perturbed in the hearts from Wdfy3-deficient mice. No alteration of autophagy was detected in the hearts from Wdfy3-deficient mice. CONCLUSION Taken together, our data suggest that Wdfy3 plays an essential role in cardiac development, which may be mediated by modulation of Notch1 signalling.
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Affiliation(s)
- Shasha Zhang
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Zongpei Song
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Lin An
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Xiaoyun Liu
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Xiao-Wen Hu
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Amber Naz
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Rujiang Zhou
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Xizhi Guo
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Lin He
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
| | - Hongxin Zhu
- Bio-X-Renji Hospital Research Center, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, China.,Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, Shanghai, China
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17
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Yang F, Huang L, Tso A, Wang H, Cui L, Lin L, Wang X, Ren M, Fang X, Liu J, Han Z, Chen J, Ouyang K. Inositol 1,4,5-trisphosphate receptors are essential for fetal-maternal connection and embryo viability. PLoS Genet 2020; 16:e1008739. [PMID: 32320395 PMCID: PMC7176088 DOI: 10.1371/journal.pgen.1008739] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Accepted: 03/25/2020] [Indexed: 01/28/2023] Open
Abstract
Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the ER membrane, which in mammals consist of 3 different subtypes (IP3R1, IP3R2, and IP3R3) encoded by 3 genes, Itpr1, Itpr2, and Itpr3, respectively. Studies utilizing genetic knockout mouse models have demonstrated that IP3Rs are essential for embryonic survival in a redundant manner. Deletion of both IP3R1 and IP3R2 has been shown to cause cardiovascular defects and embryonic lethality. However, it remains unknown which cell types account for the cardiovascular defects in IP3R1 and IP3R2 double knockout (DKO) mice. In this study, we generated conditional IP3R1 and IP3R2 knockout mouse models with both genes deleted in specific cardiovascular cell lineages. Our results revealed that deletion of IP3R1 and IP3R2 in cardiomyocytes by TnT-Cre, in endothelial / hematopoietic cells by Tie2-Cre and Flk1-Cre, or in early precursors of the cardiovascular lineages by Mesp1-Cre, resulted in no phenotypes. This demonstrated that deletion of both IP3R genes in cardiovascular cell lineages cannot account for the cardiovascular defects and embryonic lethality observed in DKO mice. We then revisited and performed more detailed phenotypic analysis in DKO embryos, and found that DKO embryos developed cardiovascular defects including reduced size of aortas, enlarged cardiac chambers, as well as growth retardation at embryonic day (E) 9.5, but in varied degrees of severity. Interestingly, we also observed allantoic-placental defects including reduced sizes of umbilical vessels and reduced depth of placental labyrinth in DKO embryos, which could occur independently from other phenotypes in DKO embryos even without obvious growth retardation. Furthermore, deletion of both IP3R1 and IP3R2 by the epiblast-specific Meox2-Cre, which targets all the fetal tissues and extraembryonic mesoderm but not extraembryonic trophoblast cells, also resulted in embryonic lethality and similar allantoic-placental defects. Taken together, our results demonstrated that IP3R1 and IP3R2 play an essential and redundant role in maintaining the integrity of fetal-maternal connection and embryonic viability.
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Affiliation(s)
- Feili Yang
- School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Lei Huang
- Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, Shenzhen, China
| | - Alexandria Tso
- University of California San Diego, School of Medicine, Department of Medicine, La Jolla, CA, United States of America
| | - Hong Wang
- School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China
| | - Li Cui
- University of California San Diego, School of Medicine, Department of Medicine, La Jolla, CA, United States of America
| | - Lizhu Lin
- University of California San Diego, School of Medicine, Department of Medicine, La Jolla, CA, United States of America
| | - Xiaohong Wang
- Department of Pharmacology, Tianjin Key Laboratory of Inflammation Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China
| | - Mingming Ren
- Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, Shenzhen, China
| | - Xi Fang
- University of California San Diego, School of Medicine, Department of Medicine, La Jolla, CA, United States of America
| | - Jie Liu
- Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen, China
| | - Zhen Han
- Department of Cardiovascular Surgery, Peking University Shenzhen Hospital, Shenzhen, China
- * E-mail: (ZH); (JC); (KO)
| | - Ju Chen
- University of California San Diego, School of Medicine, Department of Medicine, La Jolla, CA, United States of America
- * E-mail: (ZH); (JC); (KO)
| | - Kunfu Ouyang
- School of Chemical Biology and Biotechnology, State Key Laboratory of Chemical Oncogenomics, Peking University Shenzhen Graduate School, Shenzhen, China
- * E-mail: (ZH); (JC); (KO)
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18
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Liko D, Mitchell L, Campbell KJ, Ridgway RA, Jones C, Dudek K, King A, Bryson S, Stevenson D, Blyth K, Strathdee D, Morton JP, Bird TG, Knight JRP, Willis AE, Sansom OJ. Brf1 loss and not overexpression disrupts tissues homeostasis in the intestine, liver and pancreas. Cell Death Differ 2019; 26:2535-2550. [PMID: 30858608 PMCID: PMC6861133 DOI: 10.1038/s41418-019-0316-7] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2018] [Revised: 01/18/2019] [Accepted: 02/13/2019] [Indexed: 12/22/2022] Open
Abstract
RNA polymerase III (Pol-III) transcribes tRNAs and other small RNAs essential for protein synthesis and cell growth. Pol-III is deregulated during carcinogenesis; however, its role in vivo has not been studied. To address this issue, we manipulated levels of Brf1, a Pol-III transcription factor that is essential for recruitment of Pol-III holoenzyme at tRNA genes in vivo. Knockout of Brf1 led to embryonic lethality at blastocyst stage. In contrast, heterozygous Brf1 mice were viable, fertile and of a normal size. Conditional deletion of Brf1 in gastrointestinal epithelial tissues, intestine, liver and pancreas, was incompatible with organ homeostasis. Deletion of Brf1 in adult intestine and liver induced apoptosis. However, Brf1 heterozygosity neither had gross effects in these epithelia nor did it modify tumorigenesis in the intestine or pancreas. Overexpression of BRF1 rescued the phenotypes of Brf1 deletion in intestine and liver but was unable to initiate tumorigenesis. Thus, Brf1 and Pol-III activity are absolutely essential for normal homeostasis during development and in adult epithelia. However, Brf1 overexpression or heterozygosity are unable to modify tumorigenesis, suggesting a permissive, but not driving role for Brf1 in the development of epithelial cancers of the pancreas and gut.
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Affiliation(s)
- Dritan Liko
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Louise Mitchell
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Kirsteen J Campbell
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Rachel A Ridgway
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Carolyn Jones
- MRC Toxicology Unit, Hodgkin Building Lancaster Road, Leicester, LE1 9HN, UK
| | - Kate Dudek
- MRC Toxicology Unit, Hodgkin Building Lancaster Road, Leicester, LE1 9HN, UK
| | - Ayala King
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Sheila Bryson
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - David Stevenson
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Karen Blyth
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
- Institute of Cancer Sciences, University of Glasgow, Glasgow, G61 1BD, UK
| | - Douglas Strathdee
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - Jennifer P Morton
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
- Institute of Cancer Sciences, University of Glasgow, Glasgow, G61 1BD, UK
| | - Thomas G Bird
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK
| | - John R P Knight
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
| | - Anne E Willis
- MRC Toxicology Unit, Hodgkin Building Lancaster Road, Leicester, LE1 9HN, UK
| | - Owen J Sansom
- CRUK Beatson Institute, Garscube Estate, Switchback Road, Glasgow, G61 1BD, UK.
- Institute of Cancer Sciences, University of Glasgow, Glasgow, G61 1BD, UK.
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19
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Hemberger M, Hanna CW, Dean W. Mechanisms of early placental development in mouse and humans. Nat Rev Genet 2019; 21:27-43. [PMID: 31534202 DOI: 10.1038/s41576-019-0169-4] [Citation(s) in RCA: 303] [Impact Index Per Article: 50.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/09/2019] [Indexed: 02/08/2023]
Abstract
The importance of the placenta in supporting mammalian development has long been recognized, but our knowledge of the molecular, genetic and epigenetic requirements that underpin normal placentation has remained remarkably under-appreciated. Both the in vivo mouse model and in vitro-derived murine trophoblast stem cells have been invaluable research tools for gaining insights into these aspects of placental development and function, with recent studies starting to reshape our view of how a unique epigenetic environment contributes to trophoblast differentiation and placenta formation. These advances, together with recent successes in deriving human trophoblast stem cells, open up new and exciting prospects in basic and clinical settings that will help deepen our understanding of placental development and associated disorders of pregnancy.
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Affiliation(s)
- Myriam Hemberger
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada. .,Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Canada. .,Department of Medical Genetics, Cumming School of Medicine, University of Calgary, Calgary, Canada. .,Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK. .,Centre for Trophoblast Research, University of Cambridge, Cambridge, UK.
| | - Courtney W Hanna
- Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK.,Centre for Trophoblast Research, University of Cambridge, Cambridge, UK
| | - Wendy Dean
- Department of Biochemistry & Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, Canada. .,Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Canada. .,Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, UK. .,Department of Cell Biology and Anatomy, Cumming School of Medicine, University of Calgary, Calgary, Canada.
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20
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Siva plays a critical role in mouse embryonic development. Cell Death Differ 2019; 27:297-309. [PMID: 31164717 DOI: 10.1038/s41418-019-0358-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2018] [Revised: 04/14/2019] [Accepted: 05/07/2019] [Indexed: 01/05/2023] Open
Abstract
The Siva protein, named after the Hindu God of Destruction, plays important roles in apoptosis in various contexts, including downstream of death receptor activation or p53 tumor suppressor engagement. The function of Siva in organismal development and homeostasis, however, has remained uncharacterized. Here, we generate Siva knockout mice to characterize the physiological function of Siva in vivo. Interestingly, we find that Siva deficiency causes early embryonic lethality accompanied by multiple phenotypes, including developmental delay, abnormal neural tube closure, and defective placenta and yolk sac formation. Examination of Siva expression during embryogenesis shows that Siva is expressed in both embryonic and extra-embryonic tissues, including within the mesoderm, which may explain the vascular defects observed in the placenta and yolk sac. The embryonic phenotypes caused by Siva loss are not rescued by p53 deficiency, nor do they resemble those of p53 null embryos, suggesting that the embryonic function of Siva is not related to the p53 pathway. Moreover, loss of the Ripk3 necroptosis protein does not rescue the observed lethality or developmental defects, suggesting that Siva may play a non-apoptotic role in development. Collectively, these studies reveal a key role for Siva in proper embryonic development.
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21
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Scott JR. Reproductive immunology from the perspective of the clinician. J Reprod Immunol 2019; 133:27-29. [PMID: 31174079 DOI: 10.1016/j.jri.2019.05.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 05/02/2019] [Accepted: 05/24/2019] [Indexed: 11/17/2022]
Abstract
Investigators generate new and innovative ideas needed to advance knowledge, while physicians want proven treatments that provide the best care for their patients. Along with advances in reproductive immunology research, there have also been controversies such as immunologic treatments for recurrent pregnancy loss. Research deficiencies that are wasteful and misleading include: over-interpretation and extrapolation from animal studies to the human, inadequate sample sizes, lack of appropriate control groups, use of surrogate markers, associations presented as causation, un-blinded testing and treatments, unreproducible results, and non-standardized outcomes. The purpose of the EQUATOR Network (Enhancing the QUAlity Of health Research) is to improve the quality of research and its publication. These guidelines (CONSORT, STROBE, PRISMA, STARD, ARRIVE) have been accepted as mandatory by virtually all major medical journals, and all investigators should prospectively incorporate them into their study designs. From the perspective of a clinician-scientist and an editor, my premise is that the purpose of much basic science research and all clinical research is to improve the medical care of patients. Unproven and costly diagnostic tests and treatments for potential immunologic clinical problems can no longer be justified. The primary and most important outcome that should be reported for all pregnancy-related immunologic studies is the live birth rate of a healthy infant. Today's clinicians and patients expect unbiased research that leads to evidence-based recommendations for practical and effective treatments.
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Affiliation(s)
- James R Scott
- Department of Obstetrics and Gynecology, University of Iowa Carver College of Medicine, United States; University of Utah School of Medicine, Salt Lake City, Utah, 8412, United States.
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22
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Chitwood JL, Burruel VR, Halstead MM, Meyers SA, Ross PJ. Transcriptome profiling of individual rhesus macaque oocytes and preimplantation embryos. Biol Reprod 2018; 97:353-364. [PMID: 29025079 DOI: 10.1093/biolre/iox114] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2017] [Accepted: 09/01/2017] [Indexed: 11/12/2022] Open
Abstract
Early mammalian embryonic transcriptomes are dynamic throughout the process of preimplantation development. Cataloging of primate transcriptomics during early development has been accomplished in humans, but global characterization of transcripts is lacking in the rhesus macaque: a key model for human reproductive processes. We report here the systematic classification of individual macaque transcriptomes using RNA-Seq technology from the germinal vesicle stage oocyte through the blastocyst stage embryo. Major differences in gene expression were found between sequential stages, with the 4- to 8-cell stages showing the highest level of differential gene expression. Analysis of putative transcription factor binding sites also revealed a striking increase in key regulatory factors in 8-cell embryos, indicating a strong likelihood of embryonic genome activation occurring at this stage. Furthermore, clustering analyses of gene co-expression throughout this period resulted in distinct groups of transcripts significantly associated to the different embryo stages assayed. The sequence data provided here along with characterizations of major regulatory transcript groups present a comprehensive atlas of polyadenylated transcripts that serves as a useful resource for comparative studies of preimplantation development in humans and other species.
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Affiliation(s)
- James L Chitwood
- Department of Animal Science, University of California, Davis, California, USA
| | - Victoria R Burruel
- Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California, USA
| | - Michelle M Halstead
- Department of Animal Science, University of California, Davis, California, USA
| | - Stuart A Meyers
- Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California, USA
| | - Pablo J Ross
- Department of Animal Science, University of California, Davis, California, USA
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23
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Yang Y, Abdulhasan M, Awonuga A, Bolnick A, Puscheck EE, Rappolee DA. Hypoxic Stress Forces Adaptive and Maladaptive Placental Stress Responses in Early Pregnancy. Birth Defects Res 2018; 109:1330-1344. [PMID: 29105384 DOI: 10.1002/bdr2.1149] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2017] [Accepted: 10/07/2017] [Indexed: 12/19/2022]
Abstract
This review focuses on hypoxic stress and its effects on the placental lineage and the earliest differentiation events in mouse and human placental trophoblast stem cells (TSCs). Although the placenta is a decidual organ at the end of pregnancy, its earliest rapid growth and function at the start of pregnancy precedes and supports growth and function of the embryo. Earliest function requires that TSCs differentiate, however, "hypoxia" supports rapid growth, but not differentiation of TSCs. Most of the literature on earliest placental "hypoxia" studies used 2% oxygen which is normoxic for TSCs. Hypoxic stress happens when oxygen level drops below 2%. It decreases anabolism, proliferation, potency/stemness and increases differentiation, despite culture conditions that would sustain proliferation and potency. Thus, to study the pathogenesis due to TSC dysfunction, it is important to study hypoxic stress below 2%. Many studies have been performed using 0.5 to 1% oxygen in cultured mouse TSCs. From all these studies, a small number has examined human trophoblast lines and primary first trimester placental hypoxic stress responses in culture. Some other stress stimuli, aside from hypoxic stress, are used to elucidate common and unique aspects of hypoxic stress. The key outcomes produced by hypoxic stress are mitochondrial, anabolic, and proliferation arrest, and this is coupled with stemness loss and differentiation. Hypoxic stress can lead to depletion of stem cells and miscarriage, or can lead to later dysfunctions in placentation and fetal development. Birth Defects Research 109:1330-1344, 2017. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Yu Yang
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan.,Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan
| | - Mohammed Abdulhasan
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan
| | - Awoniyi Awonuga
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan
| | - Alan Bolnick
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan
| | - Elizabeth E Puscheck
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan
| | - Daniel A Rappolee
- CS Mott Center for Human Growth and Development, Department of Obstetrics & Gynecology, Reproductive Endocrinology and Infertility, Wayne State University School of Medicine, Detroit, Michigan.,Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan.,Institutes for Environmental Health Science, Wayne state University School of Medicine, Detroit, Michigan.,Department of Biology, University of Windsor, Windsor, ON, Canada
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24
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Role of recombination and faithfulness to partner in sex chromosome degeneration. Sci Rep 2018; 8:8978. [PMID: 29895905 PMCID: PMC5997740 DOI: 10.1038/s41598-018-27219-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2018] [Accepted: 05/24/2018] [Indexed: 11/08/2022] Open
Abstract
Sex determination in mammals is strongly linked to sex chromosomes. In most cases, females possess two copies of X chromosome while males have one X and one Y chromosome. It is assumed that these chromosomes originated from a pair of homologous autosomes, which diverged when recombination between them was suppressed. However, it is still debated why the sex chromosomes stopped recombining and how this process spread out over most part of the chromosomes. To study this problem, we developed a simulation model, in which the recombination rate between the sex chromosomes can freely evolve. We found that the suppression of recombination between the X and Y is spontaneous and proceeds very quickly during the evolution of population, which leads to the degeneration of the Y in males. Interestingly, the degeneration happens only when mating pairs are unfaithful. This evolutionary strategy purifies the X chromosome from defective alleles and leads to the larger number of females than males in the population. In consequence, the reproductive potential of the whole population increases. Our results imply that both the suppression of recombination and the degeneration of Y chromosome may be associated with reproductive strategy and favoured in polygamous populations with faithless mating partners.
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25
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Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning. Sci Rep 2018; 8:5632. [PMID: 29618843 PMCID: PMC5884795 DOI: 10.1038/s41598-018-24014-w] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2017] [Accepted: 03/22/2018] [Indexed: 01/23/2023] Open
Abstract
Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFβ signaling. Since Rudhira is also expressed in endothelial cells (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.
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26
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Blastocyst-Derived Stem Cell Populations under Stress: Impact of Nutrition and Metabolism on Stem Cell Potency Loss and Miscarriage. Stem Cell Rev Rep 2018; 13:454-464. [PMID: 28425063 DOI: 10.1007/s12015-017-9734-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Data from in vitro and in vivo models suggest that malnutrition and stress trigger adaptive responses, leading to small for gestational age (SGA) blastocysts with fewer cell numbers. These stress responses are initially adaptive, but become maladaptive with increasing stress exposures. The common stress responses of the blastocyst-derived stem cells, pluripotent embryonic and multipotent placental trophoblast stem cells (ESCs and TSCs), are decreased growth and potency, and increased, imbalanced and irreversible differentiation. SGA embryos may fail to produce sufficient antiluteolytic placental hormone to maintain corpus luteum progesterone secretion that provides nutrition at the implantation site. Myriad stress inputs for the stem cells in the embryo can occur in vitro during in vitro fertilization/assisted reproductive technology (IVF/ART) or in vivo. Paradoxically, stresses that diminish stem cell growth lead to a higher level of differentiation simultaneously which further decreases ESC or TSC numbers in an attempt to functionally compensate for fewer cells. In addition, prolonged or strong stress can cause irreversible differentiation. Resultant stem cell depletion is proposed as a cause of miscarriage via a "quiet" death of an ostensibly adaptive response of stem cells instead of a reactive, violent loss of stem cells or their differentiated progenies.
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27
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Jessop P, Ruzov A, Gering M. Developmental Functions of the Dynamic DNA Methylome and Hydroxymethylome in the Mouse and Zebrafish: Similarities and Differences. Front Cell Dev Biol 2018; 6:27. [PMID: 29616219 PMCID: PMC5869911 DOI: 10.3389/fcell.2018.00027] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2017] [Accepted: 03/05/2018] [Indexed: 12/25/2022] Open
Abstract
5-methylcytosine (5mC) is the best understood DNA modification and is generally believed to be associated with repression of gene expression. Over the last decade, sequentially oxidized forms of 5mC (oxi-mCs) have been discovered within the genomes of vertebrates. Their discovery was accompanied by that of the ten-eleven translocation (TET) methylcytosine dioxygenases, the enzymes that catalyze the formation of the oxi-mCs. Although a number of studies performed on different vertebrate models and embryonic stem cells demonstrated that both TET enzymes and oxi-mCs are likely to be important for several developmental processes it is currently unclear whether their developmental roles are conserved among vertebrates. Here, we summarize recent developments in this field suggesting that biological roles of TETs/oxi-mCs may significantly differ between mice and zebrafish. Thus, although the role of TET proteins in late organogenesis has been documented for both these systems; unlike in mice the enzymatic oxidation of 5mC does not seem to be involved in zygotic reprogramming or gastrulation in zebrafish. Our analysis may provide an insight into the general principles of epigenetic regulation of animal development and cellular differentiation.
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Affiliation(s)
- Peter Jessop
- School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
| | - Alexey Ruzov
- Division of Cancer and Stem Cells, Centre for Biomolecular Sciences, School of Medicine, University of Nottingham, Nottingham, United Kingdom
| | - Martin Gering
- School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, United Kingdom
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28
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Abstract
Large-scale phenotyping efforts have demonstrated that approximately 25-30% of mouse gene knockouts cause intra-uterine lethality. Analysis of these mutants has largely focussed on the embryo but not the placenta, despite the critical role of this extra-embryonic organ for developmental progression. Here, we screened 103 embryonic lethal and subviable mouse knockout lines from the Deciphering the Mechanisms of Developmental Disorders programme (https://dmdd.org.uk) for placental phenotypes. 68% of lines that are lethal at or after mid-gestation exhibited placental dys-morphologies. Early lethality (E9.5-E14.5) is almost always associated with severe placental malformations. Placental defects strongly correlate with abnormal brain, heart and vascular development. Analysis of mutant trophoblast stem cells and conditional knockouts suggests primary gene function in trophoblast for a significant number of factors that cause embryonic lethality when ablated. Our data highlight the hugely under-appreciated importance of placental defects in contributing to abnormal embryo development and suggest key molecular nodes governing placentation.
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29
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Nikolaou S, Hadjikypri X, Ioannou G, Elia A, Georgiades P. Functional and phenotypic distinction of the first two trophoblast subdivisions and identification of the border between them during early postimplantation: A prerequisite for understanding early patterning during placentogenesis. Biochem Biophys Res Commun 2018; 496:64-69. [PMID: 29305264 DOI: 10.1016/j.bbrc.2017.12.167] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2017] [Accepted: 12/30/2017] [Indexed: 12/22/2022]
Abstract
The early stages of mouse placentogenesis (placenta formation) involve poorly understood patterning events within polar trophectoderm-derived trophoblast, the progenitor of all placental trophoblast cell types. By early postimplantation [embryonic day 5.5 (E5.5)], this patterning causes early trophoblast to become subdivided into extraembryonic ectoderm (ExE) and ectoplacental cone (EPC). A prerequisite to understanding this patterning requires knowing the location of ExE-EPC border and being able to distinguish the entire ExE from EPC at E5.5/E6.5, a time when the proamnioitic cavity within ExE is not fully established. However, these issues are unknown, as they have not been directly addressed. Here, we directly addressed these using trophoblast explant culture to functionally test for the location of ExE-EPC border, combined with phenotypic characterization of trophoblast proximal and distal to it. We show for the first time that the proximal-distal level of ExE-EPC border within E5.5/E6.5 trophoblast coincides with where Reichert's membrane (outermost basement membrane of conceptus) inserts into early trophoblast and with the proximal limit of extraembryonic visceral endoderm (primitive endoderm derivative covering part of early trophoblast). Based on these novel findings, we discovered that (a) the entire E5.5/E6.5 ExE can be distinguished from EPC because it is epithelial and specifically expresses Erf and Claudin4 and (b) at E5.5/E6.5, the entire EPC differs from ExE in that it is not epithelial and specifically expresses Snail. This work is expected to contribute to understanding the cellular and molecular basis of early trophoblast patterning during placentogenesis.
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Affiliation(s)
- Stavros Nikolaou
- Department of Biological Sciences, University of Cyprus, University Campus, P.O. Box 20537, 1678 Nicosia, Cyprus
| | - Xenia Hadjikypri
- Department of Biological Sciences, University of Cyprus, University Campus, P.O. Box 20537, 1678 Nicosia, Cyprus
| | - Giasemia Ioannou
- Department of Biological Sciences, University of Cyprus, University Campus, P.O. Box 20537, 1678 Nicosia, Cyprus
| | - Artemis Elia
- Department of Biological Sciences, University of Cyprus, University Campus, P.O. Box 20537, 1678 Nicosia, Cyprus
| | - Pantelis Georgiades
- Department of Biological Sciences, University of Cyprus, University Campus, P.O. Box 20537, 1678 Nicosia, Cyprus.
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30
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High-speed, high-frequency ultrasound, in utero vector-flow imaging of mouse embryos. Sci Rep 2017; 7:16658. [PMID: 29192281 PMCID: PMC5709407 DOI: 10.1038/s41598-017-16933-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2017] [Accepted: 11/19/2017] [Indexed: 01/16/2023] Open
Abstract
Real-time imaging of the embryonic murine cardiovascular system is challenging due to the small size of the mouse embryo and rapid heart rate. High-frequency, linear-array ultrasound systems designed for small-animal imaging provide high-frame-rate and Doppler modes but are limited in regards to the field of view that can be imaged at fine-temporal and -spatial resolution. Here, a plane-wave imaging method was used to obtain high-speed image data from in utero mouse embryos and multi-angle, vector-flow algorithms were applied to the data to provide information on blood flow patterns in major organs. An 18-MHz linear array was used to acquire plane-wave data at absolute frame rates ≥10 kHz using a set of fixed transmission angles. After beamforming, vector-flow processing and image compounding, effective frame rates were on the order of 2 kHz. Data were acquired from the embryonic liver, heart and umbilical cord. Vector-flow results clearly revealed the complex nature of blood-flow patterns in the embryo with fine-temporal and -spatial resolution.
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31
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Woods L, Perez-Garcia V, Kieckbusch J, Wang X, DeMayo F, Colucci F, Hemberger M. Decidualisation and placentation defects are a major cause of age-related reproductive decline. Nat Commun 2017; 8:352. [PMID: 28874785 PMCID: PMC5585348 DOI: 10.1038/s41467-017-00308-x] [Citation(s) in RCA: 103] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Accepted: 06/20/2017] [Indexed: 12/19/2022] Open
Abstract
Mammalian reproductive performance declines rapidly with advanced maternal age. This effect is largely attributed to the exponential increase in chromosome segregation errors in the oocyte with age. Yet many pregnancy complications and birth defects that become more frequent in older mothers, in both humans and mice, occur in the absence of karyotypic abnormalities. Here, we report that abnormal embryonic development in aged female mice is associated with severe placentation defects, which result from major deficits in the decidualisation response of the uterine stroma. This problem is rooted in a blunted hormonal responsiveness of the ageing uterus. Importantly, a young uterine environment can restore normal placental as well as embryonic development. Our data highlight the pivotal, albeit under-appreciated, impact of maternal age on uterine adaptability to pregnancy as major contributor to the decline in reproductive success in older females.Advanced maternal age has been associated with lower reproductive success and higher risk of pregnancy complications. Here the authors show that maternal ageing-related embryonic abnormalities in mouse are caused by decidualisation and placentation defects that can be rescued by transferring the embryo from an old to a young uterus.
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Affiliation(s)
- Laura Woods
- Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK
- Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK
| | - Vicente Perez-Garcia
- Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK
- Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK
| | - Jens Kieckbusch
- Department of Obstetrics and Gynaecology, University of Cambridge School of Clinical Medicine, NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Box 111, Hills Road, Cambridge, CB2 0SP, UK
| | - Xiaoqiu Wang
- Reproductive and Developmental Biology Laboratory, NIEHS, Research Triangle Park, Durham, NC, 27709, USA
| | - Francesco DeMayo
- Reproductive and Developmental Biology Laboratory, NIEHS, Research Triangle Park, Durham, NC, 27709, USA
| | - Francesco Colucci
- Department of Obstetrics and Gynaecology, University of Cambridge School of Clinical Medicine, NIHR Cambridge Biomedical Research Centre, Addenbrooke's Hospital, Box 111, Hills Road, Cambridge, CB2 0SP, UK
| | - Myriam Hemberger
- Epigenetics Programme, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT, UK.
- Centre for Trophoblast Research, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK.
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32
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Saltel F, Giese A, Azzi L, Elatmani H, Costet P, Ezzoukhry Z, Dugot-Senant N, Miquerol L, Boussadia O, Wodrich H, Dubus P, Jacquemin-Sablon H. Unr defines a novel class of nucleoplasmic reticulum involved in mRNA translation. J Cell Sci 2017; 130:1796-1808. [PMID: 28386023 DOI: 10.1242/jcs.198697] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2016] [Accepted: 03/31/2017] [Indexed: 12/23/2022] Open
Abstract
Unr (officially known as CSDE1) is a cytoplasmic RNA-binding protein with roles in the regulation of mRNA stability and translation. In this study, we identified a novel function for Unr, which acts as a positive regulator of placental development. Unr expression studies in the developing placenta revealed the presence of Unr-rich foci that are apparently located in the nuclei of trophoblast giant cells (TGCs). We determined that what we initially thought to be foci, were actually cross sections of a network of double-wall nuclear membrane invaginations that contain a cytoplasmic core related to the nucleoplasmic reticulum (NR). We named them, accordingly, Unr-NRs. Unr-NRs constitute a novel type of NR because they contain high levels of poly(A) RNA and translation factors, and are sites of active translation. In murine tissues, Unr-NRs are only found in two polyploid cell types, in TGCs and hepatocytes. In vitro, their formation is linked to stress and polyploidy because, in three cancer cell lines, cytotoxic drugs that are known to promote polyploidization induce their formation. Finally, we show that Unr is required in vivo for the formation of Unr-containing NRs because these structures are absent in Unr-null TGCs.
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Affiliation(s)
- Frédéric Saltel
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France .,University of Bordeaux, F-33000 Bordeaux, France
| | - Alban Giese
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.,University of Bordeaux, F-33000 Bordeaux, France
| | - Lamia Azzi
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.,University of Bordeaux, F-33000 Bordeaux, France.,Department of Tumor Biology, CHU, F-33000 Bordeaux, France
| | - Habiba Elatmani
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.,University of Bordeaux, F-33000 Bordeaux, France
| | - Pierre Costet
- Laboratoire de Transgenèse, Université Bordeaux, F-33000 Bordeaux, France
| | - Zakaria Ezzoukhry
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.,University of Bordeaux, F-33000 Bordeaux, France
| | | | - Lucile Miquerol
- Aix-Marseille University, CNRS, IBDM UMR 7288, Marseille, France
| | | | - Harald Wodrich
- University of Bordeaux, F-33000 Bordeaux, France.,MFP CNRS UMR 5234, Microbiologie Fondamentale et Pathogénicité, Université de Bordeaux, F-33000 Bordeaux, France
| | - Pierre Dubus
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France.,University of Bordeaux, F-33000 Bordeaux, France.,Department of Tumor Biology, CHU, F-33000 Bordeaux, France
| | - Hélène Jacquemin-Sablon
- INSERM UMR1053 Bordeaux Research In Translational Oncology, BaRITOn, F-33000 Bordeaux, France .,University of Bordeaux, F-33000 Bordeaux, France
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33
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Nair RR, Kerätär JM, Autio KJ, Masud AJ, Finnilä MA, Autio-Harmainen HI, Miinalainen IJ, Nieminen PA, Hiltunen JK, Kastaniotis AJ. Genetic modifications of Mecr reveal a role for mitochondrial 2-enoyl-CoA/ACP reductase in placental development in mice. Hum Mol Genet 2017; 26:2104-2117. [DOI: 10.1093/hmg/ddx105] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2016] [Accepted: 03/13/2017] [Indexed: 01/23/2023] Open
Affiliation(s)
- Remya R. Nair
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland
| | - Juha M. Kerätär
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland
| | - Kaija J. Autio
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland
| | - Ali J. Masud
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland
| | - Mikko A.J. Finnilä
- Department of Applied Physics, University of Eastern Finland, FI-70211 Kuopio, Finland
- Research Unit of Medical Imaging, Physics and Technology, Faculty of Medicine, University of Oulu, FI-90014 Oulu, Finland
| | - Helena I. Autio-Harmainen
- Department of Pathology and Medical Research Center Oulu, Oulu University Hospital, FI-90220 Oulu, Finland
| | - Ilkka J. Miinalainen
- Electron Microscopy Core Facility, Biocenter Oulu, University of Oulu, FI-90014 Oulu, Finland
| | - Pentti A. Nieminen
- Medical Informatics and Statistics Research group, University of Oulu, FI-90014 Oulu, Finland
| | - J. Kalervo Hiltunen
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland
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34
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Kokoszka JE, Waymire KG, Flierl A, Sweeney KM, Angelin A, MacGregor GR, Wallace DC. Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction. BIOCHIMICA ET BIOPHYSICA ACTA 2016; 1857:1203-1212. [PMID: 27048932 PMCID: PMC5100012 DOI: 10.1016/j.bbabio.2016.03.026] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/25/2016] [Revised: 03/17/2016] [Accepted: 03/18/2016] [Indexed: 01/05/2023]
Abstract
The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.
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MESH Headings
- Adenine/metabolism
- Adenine Nucleotide Translocator 2/deficiency
- Adenine Nucleotide Translocator 2/genetics
- Animals
- Biological Transport
- Cell Proliferation
- Embryo, Mammalian
- Female
- Gene Expression Regulation, Developmental
- Genes, Lethal
- Heart Defects, Congenital/embryology
- Heart Defects, Congenital/genetics
- Heart Defects, Congenital/metabolism
- Heart Defects, Congenital/pathology
- Heart Failure/embryology
- Heart Failure/genetics
- Heart Failure/metabolism
- Heart Failure/pathology
- Heart Ventricles/abnormalities
- Heart Ventricles/embryology
- Heart Ventricles/metabolism
- Integrases
- Male
- Mice
- Mice, Transgenic
- Mitochondria/metabolism
- Mitochondria/pathology
- Mitochondrial Swelling/genetics
- Myocytes, Cardiac/metabolism
- Myocytes, Cardiac/pathology
- Organogenesis
- Phenotype
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Affiliation(s)
- Jason E Kokoszka
- Forensic Biology Section, Alabama Department of Forensic Sciences, Annex C, Mobile, AL 36617, United States
| | - Katrina G Waymire
- Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, United States
| | - Adrian Flierl
- The Parkinson's Institute, Sunnyvale, CA 94085, United States
| | - Katelyn M Sweeney
- Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Alessia Angelin
- Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States
| | - Grant R MacGregor
- Department of Developmental and Cell Biology, University of California Irvine, Irvine, CA 92697-2300, United States
| | - Douglas C Wallace
- Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States.
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Isaac SM, Qu D, Adamson SL. Effect of selective fetectomy on morphology of the mouse placenta. Placenta 2016; 46:11-17. [PMID: 27697216 DOI: 10.1016/j.placenta.2016.07.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2015] [Revised: 06/28/2016] [Accepted: 07/05/2016] [Indexed: 12/21/2022]
Abstract
INTRODUCTION Placental examination is recommended when genetic mutations cause fetal lethality in mice. But how fetal death alters histomorphology of the surviving mouse placenta is not known. METHODS Placentas were examined at E17.5 after fetectomy of 1-2 fetal mice per pregnancy at either embryonic day (E) 15.5 (N = 8; Fx-2 group) or E13.5 (N = 5; Fx-4 group), which left 12 ± 2 surviving fetuses per litter. RESULTS Fetectomy caused no changes in placental weights and no increases in placental hypoxia (pimonidazole staining). The size and cell morphology of the decidua and junctional zone regions were unchanged and, in the Fx-2 group, these regions became significantly less hypoxic. Significant changes in labyrinth volume included a 30% increase in the Fx-2 group and in both groups, a >50% decrease in % fetal blood space and >40% increase in % labyrinth tissue. Maternal blood sinusoid volume was unchanged. Cell death in the labyrinth was significantly increased (22-fold increase in TUNEL staining) whereas placental mRNA expression of the proliferation marker Mki67 was unchanged. mRNA expression of sFlt1 and Prl3b1 (mPL-II) was unchanged in the labyrinth and junctional zone tissues in the Fx-2 group and in whole placental tissue in the Fx-4 group. DISCUSSION Placental examination of the junctional zone and decidual regions after spontaneous fetal death in late gestation is likely to yield useful phenotypic information and abnormalities that may contribute to fetal death. In contrast, labyrinth abnormalities including increased tissue volume and reduced fetoplacental vascularity may not be due to genetic perturbation nor predate fetal death.
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Affiliation(s)
- Sarah M Isaac
- Departments of Physiology, and Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
| | - Dawei Qu
- Departments of Physiology, and Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada
| | - S Lee Adamson
- Departments of Physiology, and Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada; Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
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Phosphoinositide 3-Kinase (PI3K) Subunit p110δ Is Essential for Trophoblast Cell Differentiation and Placental Development in Mouse. Sci Rep 2016; 6:28201. [PMID: 27306493 PMCID: PMC4910077 DOI: 10.1038/srep28201] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2015] [Accepted: 05/31/2016] [Indexed: 12/24/2022] Open
Abstract
Maternal PI3K p110δ has been implicated in smaller litter sizes in mice, but its underlying mechanism remains unclear. The placenta is an indispensable chimeric organ that supports mammalian embryonic development. Using a mouse model of genetic inactivation of PI3K p110δ (p110δD910A/D910A), we show that fetuses carried by p110δD910A/D910A females were growth retarded and showed increased mortality in utero mainly during placentation. The placentas in p110δD910A/D910A females were anomalously anemic, exhibited thinner spongiotrophoblast layer and looser labyrinth zone, which indicate defective placental vasculogenesis. In addition, p110δ was detected in primary trophoblast giant cells (P-TGC) at early placentation. Maternal PI3K p110δ inactivation affected normal TGCs generation and expansion, impeded the branching of chorioallantoic placenta but enhanced the expression of matrix metalloproteinases (MMP-2, MMP-12). Poor vasculature support for the developing fetoplacental unit resulted in fetal death or gross growth retardation. These data, taken together, provide the first in vivo evidence that p110δ may play an important role in placental vascularization through manipulating trophoblast giant cell.
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Frame JM, Fegan KH, Conway SJ, McGrath KE, Palis J. Definitive Hematopoiesis in the Yolk Sac Emerges from Wnt-Responsive Hemogenic Endothelium Independently of Circulation and Arterial Identity. Stem Cells 2016; 34:431-44. [PMID: 26418893 PMCID: PMC4755868 DOI: 10.1002/stem.2213] [Citation(s) in RCA: 135] [Impact Index Per Article: 15.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2015] [Revised: 08/21/2015] [Accepted: 09/04/2015] [Indexed: 12/20/2022]
Abstract
Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires β-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remain undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit(+) cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit(+) polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/β-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit(+) EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium.
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Affiliation(s)
- Jenna M. Frame
- Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA
- Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, USA
| | - Katherine H. Fegan
- Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA
| | - Simon J. Conway
- Department of Pediatrics, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA
| | - Kathleen E. McGrath
- Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA
| | - James Palis
- Department of Pediatrics, Center for Pediatric Biomedical Research, University of Rochester Medical Center, Rochester, NY, USA
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Quétier I, Marshall JJT, Spencer-Dene B, Lachmann S, Casamassima A, Franco C, Escuin S, Worrall JT, Baskaran P, Rajeeve V, Howell M, Copp AJ, Stamp G, Rosewell I, Cutillas P, Gerhardt H, Parker PJ, Cameron AJM. Knockout of the PKN Family of Rho Effector Kinases Reveals a Non-redundant Role for PKN2 in Developmental Mesoderm Expansion. Cell Rep 2016; 14:440-448. [PMID: 26774483 PMCID: PMC4733087 DOI: 10.1016/j.celrep.2015.12.049] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2015] [Revised: 11/06/2015] [Accepted: 12/07/2015] [Indexed: 11/29/2022] Open
Abstract
In animals, the protein kinase C (PKC) family has expanded into diversely regulated subgroups, including the Rho family-responsive PKN kinases. Here, we describe knockouts of all three mouse PKN isoforms and reveal that PKN2 loss results in lethality at embryonic day 10 (E10), with associated cardiovascular and morphogenetic defects. The cardiovascular phenotype was not recapitulated by conditional deletion of PKN2 in endothelial cells or the developing heart. In contrast, inducible systemic deletion of PKN2 after E7 provoked collapse of the embryonic mesoderm. Furthermore, mouse embryonic fibroblasts, which arise from the embryonic mesoderm, depend on PKN2 for proliferation and motility. These cellular defects are reflected in vivo as dependence on PKN2 for mesoderm proliferation and neural crest migration. We conclude that failure of the mesoderm to expand in the absence of PKN2 compromises cardiovascular integrity and development, resulting in lethality.
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Affiliation(s)
- Ivan Quétier
- Kinase Biology Laboratory, John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
| | - Jacqueline J T Marshall
- Protein Phosphorylation Laboratory, Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
| | | | - Sylvie Lachmann
- Protein Phosphorylation Laboratory, Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
| | - Adele Casamassima
- Protein Phosphorylation Laboratory, Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
| | - Claudio Franco
- Instituto Medicina Molecular (iMM), Faculdade de Medicina da Universidade de Lisboa, 1649-028 Lisbon, Portugal
| | - Sarah Escuin
- Newlife Birth Defects Research Centre, Institute of Child Health, University College, London WC1N 1EH, UK
| | - Joseph T Worrall
- John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
| | - Priththivika Baskaran
- Kinase Biology Laboratory, John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
| | - Vinothini Rajeeve
- John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
| | - Michael Howell
- Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
| | - Andrew J Copp
- Newlife Birth Defects Research Centre, Institute of Child Health, University College, London WC1N 1EH, UK
| | - Gordon Stamp
- Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK
| | - Ian Rosewell
- Genetic Manipulation Services, Francis Crick Institute, Clare Hall, Herts EN6 3LD, UK
| | - Pedro Cutillas
- John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK
| | - Holger Gerhardt
- Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany
| | - Peter J Parker
- Protein Phosphorylation Laboratory, Francis Crick Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK; Division of Cancer Studies, King's College London, New Hunt's House, Saint Thomas Street, London SE1 1UL, UK.
| | - Angus J M Cameron
- Kinase Biology Laboratory, John Vane Science Centre, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK.
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Chandler RL, Magnuson T. The SWI/SNF BAF-A complex is essential for neural crest development. Dev Biol 2016; 411:15-24. [PMID: 26806701 DOI: 10.1016/j.ydbio.2016.01.015] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2015] [Revised: 01/17/2016] [Accepted: 01/20/2016] [Indexed: 10/22/2022]
Abstract
Growing evidence indicates that chromatin remodeler mutations underlie the pathogenesis of human neurocristopathies or disorders that affect neural crest cells (NCCs). However, causal relationships among chromatin remodeler subunit mutations and NCC defects remain poorly understood. Here we show that homozygous loss of ARID1A-containing, SWI/SNF chromatin remodeling complexes (BAF-A) in NCCs results in embryonic lethality in mice, with mutant embryos succumbing to heart defects. Strikingly, monoallelic loss of ARID1A in NCCs led to craniofacial defects in adult mice, including shortened snouts and low set ears, and these defects were more pronounced following homozygous loss of ARID1A, with the ventral cranial bones being greatly reduced in size. Early NCC specification and expression of the BRG1 NCC target gene, PLEXINA2, occurred normally in the absence of ARID1A. Nonetheless, mutant embryos displayed incomplete conotruncal septation of the cardiac outflow tract and defects in the posterior pharyngeal arteries, culminating in persistent truncus arteriosus and agenesis of the ductus arteriosus. Consistent with this, migrating cardiac NCCs underwent apoptosis within the circumpharyngeal ridge. Our data support the notion that multiple, distinct chromatin remodeling complexes govern genetically separable events in NCC development and highlight a potential pathogenic role for NCCs in the human BAF complex disorder, Coffin-Siris Syndrome.
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Affiliation(s)
- Ronald L Chandler
- Department of Genetics, University of North Carolina at Chapel Hill, United States
| | - Terry Magnuson
- Department of Genetics, University of North Carolina at Chapel Hill, United States.
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Neural tube opening and abnormal extraembryonic membrane development in SEC23A deficient mice. Sci Rep 2015; 5:15471. [PMID: 26494538 PMCID: PMC4616029 DOI: 10.1038/srep15471] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2015] [Accepted: 09/23/2015] [Indexed: 01/14/2023] Open
Abstract
COPII (coat protein complex-II) vesicles transport proteins from the endoplasmic reticulum (ER) to the Golgi. Higher eukaryotes have two or more paralogs of most COPII components. Here we characterize mice deficient for SEC23A and studied interactions of Sec23a null allele with the previously reported Sec23b null allele. SEC23A deficiency leads to mid-embryonic lethality associated with defective development of extraembryonic membranes and neural tube opening in midbrain. Secretion defects of multiple collagen types are observed in different connective tissues, suggesting that collagens are primarily transported in SEC23A-containing vesicles in these cells. Other extracellular matrix proteins, such as fibronectin, are not affected by SEC23A deficiency. Intracellular accumulation of unsecreted proteins leads to strong induction of the unfolded protein response in collagen-producing cells. No collagen secretion defects are observed in SEC23B deficient embryos. We report that E-cadherin is a cargo that accumulates in acini of SEC23B deficient pancreas and salivary glands. Compensatory increase of one paralog is observed in the absence of the second paralog. Haploinsufficiency of the remaining Sec23 paralog on top of homozygous inactivation of the first paralog leads to earlier lethality of embryos. Our results suggest that mammalian SEC23A and SEC23B transport overlapping yet distinct spectra of cargo in vivo.
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Yang SL, Yang M, Herrlinger S, Liang C, Lai F, Chen JF. MiR-302/367 regulate neural progenitor proliferation, differentiation timing, and survival in neurulation. Dev Biol 2015; 408:140-50. [PMID: 26441343 DOI: 10.1016/j.ydbio.2015.09.020] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2015] [Revised: 09/26/2015] [Accepted: 09/26/2015] [Indexed: 11/16/2022]
Abstract
How neural progenitor cell (NPC) behaviors are temporally controlled in early developing embryos remains undefined. The in vivo functions of microRNAs (miRNAs) in early mammalian development remain largely unknown. Mir-302/367 is a miRNA cluster that encodes miR-367 and four miR-302 members (miR302a-d). We show that miR-302b is highly expressed in early neuroepithelium and its expression decline as development progresses. We generated a mir-302/367 knockout mouse model and found that deletion of mir-302/367 results in an early embryonic lethality and open neural tube defect (NTD). NPCs exhibit enhanced proliferation, precocious differentiation, and decreased cell survival in mutant embryos. Furthermore, we identified Fgf15, Cyclin D1, and D2 as direct targets of miR-302 in NPCs in vivo, and their expression is enhanced in mutant NPCs. Ectopic expression of Cyclin D1 and D2 increases NPC proliferation, while FGF19 (human ortholog of Fgf15) overexpression leads to an increase of NPC differentiation. Thus, these findings reveal essential roles of miR-302/367 in orchestrating gene expression and NPC behaviors in neurulation; they also point to miRNAs as critical genetic components associated with neural tube formation.
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Affiliation(s)
- Si-Lu Yang
- Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| | - Mei Yang
- Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| | - Stephanie Herrlinger
- Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| | - Chen Liang
- Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA
| | - Fan Lai
- Biochemistry & Molecular Biology, University of Miami, Miami, FL 33136, USA
| | - Jian-Fu Chen
- Department of Genetics, Department of Biochemistry & Molecular Biology, University of Georgia, Athens, GA 30602, USA.
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Lin C, Yon JM, Lee BJ, Kang JK, Yun YW, Nam SY. Punicalagin improves chorioallantoic and yolk sac vasculogenesis and teratogenesis of embryos induced by nicotine exposure. J Funct Foods 2015. [DOI: 10.1016/j.jff.2015.08.029] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
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Nam BY, Kim DK, Park JT, Kang HY, Paeng J, Kim S, Park J, Um JE, Oh HJ, Han SH, Yoo TH, Kang SW. Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice. Am J Physiol Renal Physiol 2015; 309:F1060-9. [PMID: 26377795 DOI: 10.1152/ajprenal.00251.2015] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2015] [Accepted: 09/15/2015] [Indexed: 12/14/2022] Open
Abstract
In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.
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Affiliation(s)
- Bo Young Nam
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Dong Ki Kim
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea
| | - Jung Tak Park
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Hye-Young Kang
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Jisun Paeng
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Seonghun Kim
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Jimin Park
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Jae Eun Um
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Hyung Jung Oh
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Seung Hyeok Han
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Tae-Hyun Yoo
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
| | - Shin-Wook Kang
- Department of Internal Medicine, College of Medicine, Severance Biomedical Science Institute, Brain Korea 21 PLUS, Yonsei University, Seoul, Republic of Korea; and
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NDR Kinases Are Essential for Somitogenesis and Cardiac Looping during Mouse Embryonic Development. PLoS One 2015; 10:e0136566. [PMID: 26305214 PMCID: PMC4549247 DOI: 10.1371/journal.pone.0136566] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2015] [Accepted: 08/05/2015] [Indexed: 01/07/2023] Open
Abstract
Studies of mammalian tissue culture cells indicate that the conserved and distinct NDR isoforms, NDR1 and NDR2, play essential cell biological roles. However, mice lacking either Ndr1 or Ndr2 alone develop normally. Here, we studied the physiological consequences of inactivating both NDR1 and NDR2 in mice, showing that the lack of both Ndr1/Ndr2 (called Ndr1/2-double null mutants) causes embryonic lethality. In support of compensatory roles for NDR1 and NDR2, total protein and activating phosphorylation levels of the remaining NDR isoform were elevated in mice lacking either Ndr1 or Ndr2. Mice retaining one single wild-type Ndr allele were viable and fertile. Ndr1/2-double null embryos displayed multiple phenotypes causing a developmental delay from embryonic day E8.5 onwards. While NDR kinases are not required for notochord formation, the somites of Ndr1/2-double null embryos were smaller, irregularly shaped and unevenly spaced along the anterior-posterior axis. Genes implicated in somitogenesis were down-regulated and the normally symmetric expression of Lunatic fringe, a component of the Notch pathway, showed a left-right bias in the last forming somite in 50% of all Ndr1/2-double null embryos. In addition, Ndr1/2-double null embryos developed a heart defect that manifests itself as pericardial edemas, obstructed heart tubes and arrest of cardiac looping. The resulting cardiac insufficiency is the likely cause of the lethality of Ndr1/2-double null embryos around E10. Taken together, we show that NDR kinases compensate for each other in vivo in mouse embryos, explaining why mice deficient for either Ndr1 or Ndr2 are viable. Ndr1/2-double null embryos show defects in somitogenesis and cardiac looping, which reveals their essential functions and shows that the NDR kinases are critically required during the early phase of organogenesis.
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Sudano MJ, Caixeta ES, Paschoal DM, Martins A, Machado R, Buratini J, Landim-Alvarenga FDC. Cryotolerance and global gene-expression patterns of Bos taurus indicus and Bos taurus taurus in vitro- and in vivo-produced blastocysts. Reprod Fertil Dev 2015; 26:1129-41. [PMID: 24025608 DOI: 10.1071/rd13099] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2013] [Accepted: 08/09/2013] [Indexed: 01/05/2023] Open
Abstract
In a 2×2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7±2.0% vs 27.0±2.0%) cows. The total numbers of ova or embryos recovered (5.5±0.9 vs 3.7±0.8) and transferable embryos (3.8±1.0 vs 2.3±0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n=75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n=89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.
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Affiliation(s)
- Mateus J Sudano
- São Paulo State University, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction and Veterinary Radiology, Rubião Jr. s/n°, 18618-970, Botucatu-SP, Brazil
| | - Ester S Caixeta
- University of José Rosário Vellano (UNIFENAS), Department of Animal Reproduction, 37130-000, Alfenas-MG, Brazil
| | - Daniela M Paschoal
- São Paulo State University, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction and Veterinary Radiology, Rubião Jr. s/n°, 18618-970, Botucatu-SP, Brazil
| | - Alicio Martins
- São Paulo State University, School of Veterinary Medicine, Department of Clinical Surgery and Animal Reproduction, 16050-680, Araçatuba-SP, Brazil
| | - Rui Machado
- Embrapa Southeast Cattle, PO Box 339, 13560-970, São Carlos-SP, Brazil
| | - José Buratini
- São Paulo State University, Department of Physiology, Rubião Jr. s/n°, 18618-970, Botucatu-SP, Brazil
| | - Fernanda D C Landim-Alvarenga
- São Paulo State University, School of Veterinary Medicine and Animal Science, Department of Animal Reproduction and Veterinary Radiology, Rubião Jr. s/n°, 18618-970, Botucatu-SP, Brazil
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Armistead J, Patel N, Wu X, Hemming R, Chowdhury B, Basra GS, Del Bigio MR, Ding H, Triggs-Raine B. Growth arrest in the ribosomopathy, Bowen-Conradi syndrome, is due to dramatically reduced cell proliferation and a defect in mitotic progression. Biochim Biophys Acta Mol Basis Dis 2015; 1852:1029-37. [PMID: 25708872 DOI: 10.1016/j.bbadis.2015.02.007] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2014] [Revised: 02/10/2015] [Accepted: 02/14/2015] [Indexed: 02/05/2023]
Abstract
Bowen-Conradi syndrome (BCS) is a ribosomopathy characterized by severe developmental delay and growth failure that typically leads to death by one year of age. It is caused by a c.257A>G, p.D86G substitution in the ribosomal biogenesis protein, Essential for Mitotic Growth 1 (EMG1). We generated a knock-in of the D86G substitution in mice to characterize the effects of EMG1 deficiency, particularly in the brain, where EMG1 expression is high. Embryos homozygous for the mutation in Emg1 were small for gestational age with neural tube defects, and died between embryonic days 8.5 and 12.5. These embryos exhibited dramatically reduced cell proliferation, which we also detected in autopsy brain tissue and bone marrow of BCS patients, consistent with a requirement for high levels of EMG1 in tissues with rapid cell proliferation. In fibroblasts derived from the BCS mouse embryos, we detected a high proportion of binucleated cells, indicating that a mitotic defect underlies the growth arrest in BCS. These studies add to growing evidence of a link between ribosome biogenesis, mitotic progression, and brain development that is currently unexplored.
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Affiliation(s)
- Joy Armistead
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada; Institute of Developmental Biology, University of Cologne, Cologne, Germany.
| | - Nehal Patel
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada.
| | - Xiaoli Wu
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada.
| | - Richard Hemming
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada.
| | - Biswajit Chowdhury
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
| | - Gagandeep Singh Basra
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada.
| | - Marc R Del Bigio
- Department of Pathology, University of Manitoba, Winnipeg, MB, Canada; Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
| | - Hao Ding
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada.
| | - Barbara Triggs-Raine
- Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, Canada; Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
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47
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Norris FC, Siow BM, Cleary JO, Wells JA, De Castro SC, Ordidge RJ, Greene ND, Copp AJ, Scambler PJ, Alexander DC, Lythgoe MF. Diffusion microscopic MRI of the mouse embryo: Protocol and practical implementation in the splotch mouse model. Magn Reson Med 2015; 73:731-9. [PMID: 24634098 PMCID: PMC4737188 DOI: 10.1002/mrm.25145] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2013] [Revised: 01/01/2014] [Accepted: 01/03/2014] [Indexed: 12/13/2022]
Abstract
PURPOSE Advanced methodologies for visualizing novel tissue contrast are essential for phenotyping the ever-increasing number of mutant mouse embryos being generated. Although diffusion microscopic MRI (μMRI) has been used to phenotype embryos, widespread routine use is limited by extended scanning times, and there is no established experimental procedure ensuring optimal data acquisition. METHODS We developed two protocols for designing experimental procedures for diffusion μMRI of mouse embryos, which take into account the effect of embryo preparation and pulse sequence parameters on resulting data. We applied our protocols to an investigation of the splotch mouse model as an example implementation. RESULTS The protocols provide DTI data in 24 min per direction at 75 μm isotropic using a three-dimensional fast spin-echo sequence, enabling preliminary imaging in 3 h (6 directions plus one unweighted measurement), or detailed imaging in 9 h (42 directions plus six unweighted measurements). Application to the splotch model enabled assessment of spinal cord pathology. CONCLUSION We present guidelines for designing diffusion μMRI experiments, which may be adapted for different studies and research facilities. As they are suitable for routine use and may be readily implemented, we hope they will be adopted by the phenotyping community.
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Affiliation(s)
- Francesca C. Norris
- UCL Centre for Advanced Biomedical Imaging, Division of MedicineUniversity College LondonLondonUnited Kingdom
- Centre for Mathematics and Physics in the Life Sciences and EXperimental Biology (CoMPLEX)University College LondonLondonUnited Kingdom
| | - Bernard M. Siow
- UCL Centre for Advanced Biomedical Imaging, Division of MedicineUniversity College LondonLondonUnited Kingdom
- Centre for Medical Image Computing, Departments of Medical Physics and Bioengineering and Computer ScienceUniversity College LondonUnited Kingdom
| | - Jon O. Cleary
- UCL Centre for Advanced Biomedical Imaging, Division of MedicineUniversity College LondonLondonUnited Kingdom
- Department of Anatomy and NeuroscienceUniversity of MelbourneAustralia
| | - Jack A. Wells
- UCL Centre for Advanced Biomedical Imaging, Division of MedicineUniversity College LondonLondonUnited Kingdom
| | - Sandra C.P. De Castro
- Neural Development Unit, UCL Institute of Child HealthUniversity College LondonLondonUnited Kingdom
| | - Roger J. Ordidge
- Department of Anatomy and NeuroscienceUniversity of MelbourneAustralia
| | - Nicholas D.E. Greene
- Neural Development Unit, UCL Institute of Child HealthUniversity College LondonLondonUnited Kingdom
| | - Andrew J. Copp
- Neural Development Unit, UCL Institute of Child HealthUniversity College LondonLondonUnited Kingdom
| | - Peter J. Scambler
- Molecular Medicine Unit, UCL Institute of Child HealthUniversity College LondonLondonUnited Kingdom
| | - Daniel. C. Alexander
- Centre for Medical Image Computing, Departments of Medical Physics and Bioengineering and Computer ScienceUniversity College LondonUnited Kingdom
| | - Mark F. Lythgoe
- UCL Centre for Advanced Biomedical Imaging, Division of MedicineUniversity College LondonLondonUnited Kingdom
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48
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Wong LF, Porter TF, Scott JR, Cochrane Pregnancy and Childbirth Group. Immunotherapy for recurrent miscarriage. Cochrane Database Syst Rev 2014; 2014:CD000112. [PMID: 25331518 PMCID: PMC7051032 DOI: 10.1002/14651858.cd000112.pub3] [Citation(s) in RCA: 50] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
Abstract
BACKGROUND Because immunological aberrations might be the cause of miscarriage in some women, several immunotherapies have been used to treat women with otherwise unexplained recurrent pregnancy loss. OBJECTIVES The objective of this review was to assess the effects of any immunotherapy, including paternal leukocyte immunization and intravenous immunoglobulin on the live birth rate in women with previous unexplained recurrent miscarriages. SEARCH METHODS We searched the Cochrane Pregnancy and Childbirth Group's Trials Register (11 February 2014) and reference lists of retrieved studies. SELECTION CRITERIA Randomized trials of immunotherapies used to treat women with three or more prior miscarriages and no more than one live birth after, in whom all recognized non-immunologic causes of recurrent miscarriage had been ruled out and no simultaneous treatment was given. DATA COLLECTION AND ANALYSIS The review author and the two co-authors independently extracted data and assessed study quality for all studies considered for this review. MAIN RESULTS Twenty trials of high quality were included. The various forms of immunotherapy did not show significant differences between treatment and control groups in terms of subsequent live births: paternal cell immunization (12 trials, 641 women), Peto odds ratio (Peto OR) 1.23, 95% confidence interval (CI) 0.89 to 1.70; third-party donor cell immunization (three trials, 156 women), Peto OR 1.39, 95% CI 0.68 to 2.82; trophoblast membrane infusion (one trial, 37 women), Peto OR 0.40, 95% CI 0.11 to 1.45; or intravenous immunoglobulin, (eight trials, 303 women), Peto OR 0.98, 95% CI 0.61 to 1.58. AUTHORS' CONCLUSIONS Paternal cell immunization, third-party donor leukocytes, trophoblast membranes, and intravenous immunoglobulin provide no significant beneficial effect over placebo in improving the live birth rate.
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Affiliation(s)
- Luchin F Wong
- Intermountain HealthcareMaternal‐Fetal MedicineMurrayUtahUSA84132
- University of Utah Medical CentreDepartment of Obstetrics and GynaecologySalt Lake CityUtahUSA
| | - T Flint Porter
- Intermountain HealthcareMaternal‐Fetal MedicineMurrayUtahUSA84132
- University of Utah Medical CentreDepartment of Obstetrics and GynaecologySalt Lake CityUtahUSA
| | - James R Scott
- University of Utah Medical CentreDepartment of Obstetrics and GynaecologySalt Lake CityUtahUSA
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Peltzer N, Rieser E, Taraborrelli L, Draber P, Darding M, Pernaute B, Shimizu Y, Sarr A, Draberova H, Montinaro A, Martinez-Barbera JP, Silke J, Rodriguez TA, Walczak H. HOIP deficiency causes embryonic lethality by aberrant TNFR1-mediated endothelial cell death. Cell Rep 2014; 9:153-165. [PMID: 25284787 DOI: 10.1016/j.celrep.2014.08.066] [Citation(s) in RCA: 206] [Impact Index Per Article: 18.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2014] [Revised: 07/31/2014] [Accepted: 08/26/2014] [Indexed: 10/25/2022] Open
Abstract
Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP's catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death.
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Affiliation(s)
- Nieves Peltzer
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Eva Rieser
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Lucia Taraborrelli
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Peter Draber
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Maurice Darding
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Barbara Pernaute
- British Heart Foundation Centre for Research Excellence, National Heart and Lung Institute (NHLI), Imperial Centre for Translational and Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK
| | - Yutaka Shimizu
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Aida Sarr
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Helena Draberova
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Antonella Montinaro
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK
| | - Juan Pedro Martinez-Barbera
- Birth Defects Research Centre, Developmental Biology and Cancer Programme, UCL Institute of Child Health, London WC1N 1EH, UK
| | - John Silke
- The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; Department of Medical Biology, University of Melbourne, Parkville, VIC 3050, Australia
| | - Tristan A Rodriguez
- British Heart Foundation Centre for Research Excellence, National Heart and Lung Institute (NHLI), Imperial Centre for Translational and Experimental Medicine, Imperial College London, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK
| | - Henning Walczak
- Centre for Cell Death, Cancer, and Inflammation (CCCI), UCL Cancer Institute, University College London, 72 Huntley Street, London WC1E 6DD, UK.
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50
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Szabo R, Peters DE, Kosa P, Camerer E, Bugge TH. Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival. PLoS Genet 2014; 10:e1004470. [PMID: 25078604 PMCID: PMC4117450 DOI: 10.1371/journal.pgen.1004470] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2014] [Accepted: 05/14/2014] [Indexed: 11/19/2022] Open
Abstract
The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival. Development of mammalian embryos is dependent on an efficient exchange of nutrients, oxygen, and waste products between the mother and the embryo. The interface between the two systems is provided by the placenta in a form of a specialized epithelium that both facilitates the transport of molecules between the mother and the embryo and screens the substances that can pass between the maternal and fetal tissues. We now show that two independent signaling pathways that include the serine proteases, matriptase and prostasin, and a G protein-coupled receptor PAR-2, are critical for the establishment of a functional feto-maternal interface by specifically regulating the barrier properties of the placental epithelium. Because aberrant formation of epithelial barriers is an underlying feature of a great variety of human developmental abnormalities, the identification of the two protease-dependent signaling pathways critical for the barrier formation in embryonic tissues may help pinpoint molecular mechanisms involved in the etiology of these conditions.
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Affiliation(s)
- Roman Szabo
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Diane E. Peters
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
- Program of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts, United States of America
| | - Peter Kosa
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Eric Camerer
- INSERM U970, Paris Cardiovascular Research Centre, Paris, France
- Université Paris-Descartes, Paris, France
| | - Thomas H. Bugge
- Proteases and Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland, United States of America
- * E-mail:
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