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1.
mBio ; 12(3): e0022721, 2021 06 29.
Article in English | MEDLINE | ID: mdl-34061599

ABSTRACT

Human cytomegalovirus (HCMV) presents a major health burden in the immunocompromised and in stem cell transplant medicine. A lack of understanding about the mechanisms of HCMV latency in undifferentiated CD34+ stem cells, and how latency is broken for the virus to enter the lytic phase of its infective cycle, has hampered the development of essential therapeutics. Using a human induced pluripotent stem cell (iPSC) model of HCMV latency and patient-derived myeloid cell progenitors, we demonstrate that bone morphogenetic protein receptor type 2 (BMPR2) is necessary for HCMV latency. In addition, we define a crucial role for the transcription factor Yin Yang 1 (YY1) in HCMV latency; high levels of YY1 are maintained in latently infected cells as a result of BMPR2 signaling through the SMAD4/SMAD6 axis. Activation of SMAD4/6, through BMPR2, inhibits TGFbeta receptor signaling, which leads to the degradation of YY1 via induction of a cellular microRNA (miRNA), hsa-miR-29a. Pharmacological targeting of BMPR2 in progenitor cells results in the degradation of YY1 and an inability to maintain latency and renders cells susceptible to T cell killing. These data argue that BMPR2 plays a role in HCMV latency and is a new potential therapeutic target for maintaining or disrupting HCMV latency in myeloid progenitors. IMPORTANCE Understanding the mechanisms which regulate HCMV latency could allow therapeutic targeting of the latent virus reservoir from where virus reactivation can cause severe disease. We show that the BMPR2/TGFbeta receptor/YY1 signaling axis is crucial to maintain HCMV latency in undifferentiated cells and that pharmacological reduction of BMPR2 in latently infected cells leads to reactivation of the viral lytic transcription program, which renders the infected cell open to immune detection and clearance in infected individuals. Therefore, this work identifies key host-virus interactions which regulate HCMV latent infection. It also demonstrates a potential new therapeutic approach to reduce HCMV reactivation-mediated disease by the treatment of donor stem cells/organs prior to transplantation, which could have a major impact in the transplant disease setting.


Subject(s)
Bone Morphogenetic Protein Receptors, Type II/metabolism , Cytomegalovirus/physiology , Host-Pathogen Interactions , Induced Pluripotent Stem Cells/virology , Myeloid Cells/virology , Signal Transduction , Virus Latency , YY1 Transcription Factor/metabolism , Bone Morphogenetic Protein Receptors, Type II/genetics , Cells, Cultured , Humans , THP-1 Cells , YY1 Transcription Factor/genetics
2.
N Biotechnol ; 63: 1-9, 2021 Jul 25.
Article in English | MEDLINE | ID: mdl-33588094

ABSTRACT

The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40-80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line.


Subject(s)
Endothelial Progenitor Cells/cytology , Stem Cell Transplantation , Cellular Reprogramming , Humans
3.
Am J Respir Cell Mol Biol ; 63(2): 160-171, 2020 08.
Article in English | MEDLINE | ID: mdl-32255665

ABSTRACT

Mutations in the gene encoding BMPR2 (bone morphogenetic protein type 2 receptor) are the major cause of heritable pulmonary arterial hypertension (PAH). Point mutations in the BMPR2 ligand-binding domain involving cysteine residues (such as C118W) are causative of PAH and predicted to cause protein misfolding. Using heterologous overexpression systems, we showed previously that these mutations lead to retention of BMPR2 in the endoplasmic reticulum but are partially rescued by chemical chaperones. Here, we sought to determine whether the chemical chaperone 4-phenylbutyrate (4PBA) restores BMPR2 signaling in primary cells and in a knockin mouse harboring a C118W mutation. First, we confirmed dysfunctional BMP signaling in dermal fibroblasts isolated from a family with PAH segregating the BMPR2 C118W mutation. After BMP4 treatment, the induction of downstream signaling targets (Smad1/5, ID1 [inhibitor of DNA binding 1], and ID2) was significantly reduced in C118W mutant cells. Treatment with 4PBA significantly rescued Smad1/5, ID1, and ID2 expression. Pulmonary artery smooth muscle cells isolated from the lungs of heterozygous mice harboring the Bmpr2 C118W mutation exhibited significantly increased proliferation. In the presence of 4PBA, hyperproliferation was dramatically reduced. Furthermore, in vivo, 4PBA treatment of Bmpr2 C118W mice partially rescued Bmpr2 expression, restored downstream signaling, and improved vascular remodeling. These findings demonstrate in primary cells and in a knockin mouse that the repurposed small-molecule chemical chaperone 4PBA might be a promising precision medicine approach to treat PAH in patients with specific subtypes of BMPR2 mutation involving cysteine substitutions in the ligand-binding domain.


Subject(s)
Bone Morphogenetic Protein Receptors, Type II/genetics , Cysteine/genetics , Mutation/genetics , Organophosphorus Compounds/pharmacology , Pulmonary Arterial Hypertension/drug therapy , Signal Transduction/drug effects , Animals , Cell Proliferation/drug effects , Cell Proliferation/genetics , Cells, Cultured , Humans , Mice , Muscle, Smooth, Vascular/drug effects , Myocytes, Smooth Muscle/drug effects , Pulmonary Arterial Hypertension/genetics , Pulmonary Artery/drug effects , Signal Transduction/genetics , Vascular Remodeling/drug effects , Vascular Remodeling/genetics
4.
Front Microbiol ; 10: 2233, 2019.
Article in English | MEDLINE | ID: mdl-31649625

ABSTRACT

Herpesviruses undergo life-long latent infection which can be life-threatening in the immunocompromised. Models of latency and reactivation of human cytomegalovirus (HCMV) include primary myeloid cells, cells known to be important for HCMV latent carriage and reactivation in vivo. However, primary cells are limited in availability, and difficult to culture and to genetically modify; all of which have hampered our ability to fully understand virus/host interactions of this persistent human pathogen. We have now used iPSCs to develop a model cell system to study HCMV latency and reactivation in different cell types after their differentiation down the myeloid lineage. Our results show that iPSCs can effectively mimic HCMV latency/reactivation in primary myeloid cells, allowing molecular interrogations of the viral latent/lytic switch. This model may also be suitable for analysis of other viruses, such as HIV and Zika, which also infect cells of the myeloid lineage.

5.
J Pathol ; 249(3): 356-367, 2019 11.
Article in English | MEDLINE | ID: mdl-31257577

ABSTRACT

Pulmonary arterial hypertension (PAH) is characterised by an increase in mean pulmonary arterial pressure which almost invariably leads to right heart failure and premature death. More than 70% of familial PAH and 20% of idiopathic PAH patients carry heterozygous mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2). However, the incomplete penetrance of BMPR2 mutations suggests that other genetic and environmental factors contribute to the disease. In the current study, we investigate the contribution of autophagy in the degradation of BMPR2 in pulmonary vascular cells. We demonstrate that endogenous BMPR2 is degraded through the lysosome in primary human pulmonary artery endothelial (PAECs) and smooth muscle cells (PASMCs): two cell types that play a key role in the pathology of the disease. By means of an elegant HaloTag system, we show that a block in lysosomal degradation leads to increased levels of BMPR2 at the plasma membrane. In addition, pharmacological or genetic manipulations of autophagy allow us to conclude that autophagy activation contributes to BMPR2 degradation. It has to be further investigated whether the role of autophagy in the degradation of BMPR2 is direct or through the modulation of the endocytic pathway. Interestingly, using an iPSC-derived endothelial cell model, our findings indicate that BMPR2 heterozygosity alone is sufficient to cause an increased autophagic flux. Besides BMPR2 heterozygosity, pro-inflammatory cytokines also contribute to an augmented autophagy in lung vascular cells. Furthermore, we demonstrate an increase in microtubule-associated protein 1 light chain 3 beta (MAP1LC3B) levels in lung sections from PAH induced in rats. Accordingly, pulmonary microvascular endothelial cells (MVECs) from end-stage idiopathic PAH patients present an elevated autophagic flux. Our findings support a model in which an increased autophagic flux in PAH patients contributes to a greater decrease in BMPR2 levels. Altogether, this study sheds light on the basic mechanisms of BMPR2 degradation and highlights a crucial role for autophagy in PAH. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Subject(s)
Autophagy , Bone Morphogenetic Protein Receptors, Type II/metabolism , Endothelial Cells/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Arterial Hypertension/metabolism , Adult , Aged , Aged, 80 and over , Animals , Arterial Pressure , Bone Morphogenetic Protein Receptors, Type II/genetics , Cell Line , Cytokines/metabolism , Disease Models, Animal , Endothelial Cells/pathology , Female , Heterozygote , Humans , Inflammation Mediators/metabolism , Lysosomes/metabolism , Lysosomes/pathology , Male , Microtubule-Associated Proteins/metabolism , Middle Aged , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Myocytes, Smooth Muscle/pathology , Proteolysis , Pulmonary Arterial Hypertension/pathology , Pulmonary Arterial Hypertension/physiopathology , Pulmonary Artery/metabolism , Pulmonary Artery/pathology , Pulmonary Artery/physiopathology , Rats , Signal Transduction , Young Adult
6.
Echo Res Pract ; 6(1): R25-R39, 2019 Mar 01.
Article in English | MEDLINE | ID: mdl-30763278

ABSTRACT

Tricuspid regurgitation natural history and treatment remains poorly understood. Right ventricular function is a key factor in determining prognosis, timing for intervention and longer-term outcome. The right ventricle is a thin walled chamber with a predominance of longitudinal fibres and a shared ventricular septum. In health, the low-pressure pulmonary circulation results in a highly compliant RV well equipped to respond to changes in preload but sensitive to even small alterations in afterload. In Part 1 of this article, discussion focuses on key principles of ventricular function assessment and the importance of right ventricular chamber size, volumes and ejection fraction, particularly in risk stratification in tricuspid regurgitation. Part 2 of this article provides an understanding of the causes of tricuspid regurgitation in the contemporary era, with emphasis on key patient groups and their management.

7.
PLoS One ; 14(1): e0211450, 2019.
Article in English | MEDLINE | ID: mdl-30703135

ABSTRACT

Although mutations in several genes have been reported in pulmonary arterial hypertension (PAH), most of PAH cases do not carry these mutations. This study aimed to identify a novel cause of PAH. To determine the disease-causing variants, direct sequencing and multiplex ligation-dependent probe amplification were performed to analyze 18 families with multiple affected family members with PAH. In one of the 18 families with PAH, no disease-causing variants were found in any of BMPR2, ACVRL1, ENG, SMAD1/4/8, BMPR1B, NOTCH3, CAV1, or KCNK3. In this family, a female proband and her paternal aunt developed PAH in their childhood. Whole-exome next-generation sequencing was performed in the 2 PAH patients and the proband's healthy mother, and a BRCA1-associated protein (BRAP) gene variant, p.Arg554Leu, was identified in the 2 family members with PAH, but not in the proband's mother without PAH. Functional analyses were performed using human pulmonary arterial smooth muscle cells (hPASMCs). Knockdown of BRAP via small interfering RNA in hPASMCs induced p53 signaling pathway activation and decreased cell proliferation. Overexpression of either wild-type BRAP or p.Arg554Leu-BRAP cDNA constructs caused cell death confounding these studies, however we observed higher levels of p53 signaling inactivation and hPASMC proliferation in cells expressing p.Arg554Leu-BRAP compared to wild-type BRAP. In addition, p.Arg554Leu-BRAP induced decreased apoptosis of hPASMCs compared with wild-type BRAP. In conclusion, we have identified a novel variant of BRAP in a Japanese family with PAH and our results suggest it could have a gain-of-function. This study sheds light on new mechanism of PAH pathogenesis.


Subject(s)
Exome/genetics , Familial Primary Pulmonary Hypertension/genetics , Familial Primary Pulmonary Hypertension/pathology , Muscle, Smooth, Vascular/pathology , Mutation , Pulmonary Artery/pathology , Ubiquitin-Protein Ligases/genetics , Adolescent , Adult , Apoptosis , Cells, Cultured , Child , Child, Preschool , Female , Humans , Infant , Male , Muscle, Smooth, Vascular/metabolism , Pedigree , Pulmonary Artery/metabolism , Signal Transduction , Exome Sequencing , Young Adult
9.
Nat Commun ; 8: 14079, 2017 01 13.
Article in English | MEDLINE | ID: mdl-28084316

ABSTRACT

Heterozygous germ-line mutations in the bone morphogenetic protein type-II receptor (BMPR-II) gene underlie heritable pulmonary arterial hypertension (HPAH). Although inflammation promotes PAH, the mechanisms by which inflammation and BMPR-II dysfunction conspire to cause disease remain unknown. Here we identify that tumour necrosis factor-α (TNFα) selectively reduces BMPR-II transcription and mediates post-translational BMPR-II cleavage via the sheddases, ADAM10 and ADAM17 in pulmonary artery smooth muscle cells (PASMCs). TNFα-mediated suppression of BMPR-II subverts BMP signalling, leading to BMP6-mediated PASMC proliferation via preferential activation of an ALK2/ACTR-IIA signalling axis. Furthermore, TNFα, via SRC family kinases, increases pro-proliferative NOTCH2 signalling in HPAH PASMCs with reduced BMPR-II expression. We confirm this signalling switch in rodent models of PAH and demonstrate that anti-TNFα immunotherapy reverses disease progression, restoring normal BMP/NOTCH signalling. Collectively, these findings identify mechanisms by which BMP and TNFα signalling contribute to disease, and suggest a tractable approach for therapeutic intervention in PAH.


Subject(s)
Bone Morphogenetic Protein Receptors, Type II/metabolism , Familial Primary Pulmonary Hypertension/metabolism , Receptor, Notch2/metabolism , Receptor, Notch3/metabolism , Tumor Necrosis Factor-alpha/metabolism , ADAM10 Protein/genetics , ADAM10 Protein/metabolism , Animals , Bone Morphogenetic Protein 6/metabolism , Bone Morphogenetic Protein Receptors, Type II/genetics , Familial Primary Pulmonary Hypertension/genetics , Female , Humans , Male , Mice , Mice, Inbred C57BL , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/metabolism , Rats , Receptor, Notch2/genetics , Receptor, Notch3/genetics , Signal Transduction , Tumor Necrosis Factor-alpha/genetics
10.
N Biotechnol ; 32(1): 191-8, 2015 Jan 25.
Article in English | MEDLINE | ID: mdl-25064145

ABSTRACT

As vertebrates proceed through embryonic development the growing organism cannot survive on diffusion of oxygen and nutrients alone and establishment of vascular system is fundamental for embryonic development to proceed. Dysfunction of the vascular system in adults is at the heart of many disease states such as hypertension and atherosclerosis. In this review we will focus on attempts to generate the key cells of the vascular system, the endothelial and smooth muscle cells, using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). Regardless of their origin, be it embryonic or via somatic cell reprogramming, pluripotent stem cells provide limitlessly self-renewing populations of material suitable for the generation of multi-lineage isogenic vascular cells-types that can be used as tools to study normal cell and tissue biology, model disease states and also as tools for drug screening and future cell therapies.


Subject(s)
Blood Vessels/physiology , Cell Differentiation , Cellular Reprogramming , Regenerative Medicine/methods , Animals , Endothelial Cells/cytology , Humans , Myocytes, Smooth Muscle/cytology
11.
J Vis Exp ; (106): e53384, 2015 Dec 23.
Article in English | MEDLINE | ID: mdl-26780290

ABSTRACT

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.


Subject(s)
Blood Cells/cytology , Cell Culture Techniques/methods , Endothelial Cells/cytology , Adult , Cellular Reprogramming , Humans , Induced Pluripotent Stem Cells/cytology
12.
PLoS One ; 9(3): e91334, 2014.
Article in English | MEDLINE | ID: mdl-24651450

ABSTRACT

The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole "HOX transcriptome" of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity.


Subject(s)
Endothelial Cells/metabolism , Gene Expression Profiling , Genes, Homeobox , Homeodomain Proteins/genetics , Adult , Animals , Cell Differentiation/genetics , Cell Line , Cluster Analysis , Embryonic Stem Cells/metabolism , Endothelial Cells/cytology , Endothelial Cells/ultrastructure , Fetus/embryology , Fetus/metabolism , Gene Ontology , Homeodomain Proteins/metabolism , Humans , Lung/cytology , Lung/embryology , Mice , Neovascularization, Physiologic/genetics , Oligonucleotide Array Sequence Analysis , Organ Specificity/genetics , Phenotype , Pulmonary Artery/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Real-Time Polymerase Chain Reaction , Reproducibility of Results
13.
Stem Cell Res Ther ; 3(4): 31, 2012 Aug 16.
Article in English | MEDLINE | ID: mdl-22897941

ABSTRACT

Ischaemia-related diseases such as peripheral artery disease and coronary heart disease constitute a major issue in medicine as they affect millions of individuals each year and represent a considerable economic burden to healthcare systems. If the underlying ischaemia is not sufficiently resolved it can lead to tissue damage, with subsequent cell death. Treating such diseases remains difficult and several strategies have been used to stimulate the growth of blood vessels and promote regeneration of ischaemic tissues, such as the use of recombinant proteins and gene therapy. Although these approaches remain promising, they have limitations and results from clinical trials using these methods have had limited success. Recently, there has been growing interest in the therapeutic potential of using a cell-based approach to treat vasodegenerative disorders. In vascular medicine, various stem cells and adult progenitors have been highlighted as having a vasoreparative role in ischaemic tissues. This review will examine the clinical potential of several stem and progenitor cells that may be utilised to regenerate defunct or damaged vasculature and restore blood flow to the ischaemic tissue. In particular, we focus on the therapeutic potential of endothelial progenitor cells as an exciting new option for the treatment of ischaemic diseases.


Subject(s)
Ischemia/therapy , Stem Cell Transplantation , Stem Cells/cytology , Adult Stem Cells/cytology , Cell- and Tissue-Based Therapy , Endothelium, Vascular/cytology , Humans , Neovascularization, Physiologic , Pluripotent Stem Cells/cytology
14.
Stem Cells Transl Med ; 1(12): 855-65, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23283547

ABSTRACT

Induced pluripotent stem cells (iPSCs) have the potential to generate patient-specific tissues for disease modeling and regenerative medicine applications. However, before iPSC technology can progress to the translational phase, several obstacles must be overcome. These include uncertainty regarding the ideal somatic cell type for reprogramming, the low kinetics and efficiency of reprogramming, and karyotype discrepancies between iPSCs and their somatic precursors. Here we describe the use of late-outgrowth endothelial progenitor cells (L-EPCs), which possess several favorable characteristics, as a cellular substrate for the generation of iPSCs. We have developed a protocol that allows the reliable isolation of L-EPCs from peripheral blood mononuclear cell preparations, including frozen samples. As a proof-of-principle for clinical applications we generated EPC-iPSCs from both healthy individuals and patients with heritable and idiopathic forms of pulmonary arterial hypertension. L-EPCs grew clonally; were highly proliferative, passageable, and bankable; and displayed higher reprogramming kinetics and efficiencies compared with dermal fibroblasts. Unlike fibroblasts, the high efficiency of L-EPC reprogramming allowed for the reliable generation of iPSCs in a 96-well format, which is compatible with high-throughput platforms. Array comparative genome hybridization analysis of L-EPCs versus donor-matched circulating monocytes demonstrated that L-EPCs have normal karyotypes compared with their subject's reference genome. In addition, >80% of EPC-iPSC lines tested did not acquire any copy number variations during reprogramming compared with their parent L-EPC line. This work identifies L-EPCs as a practical and efficient cellular substrate for iPSC generation, with the potential to address many of the factors currently limiting the translation of this technology.


Subject(s)
Adult Stem Cells/cytology , Cell Culture Techniques/methods , Endothelium, Vascular/cytology , Hematopoietic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Adult , Adult Stem Cells/physiology , Animals , Cell Differentiation/physiology , Cell Lineage/physiology , Cellular Reprogramming/physiology , Hematopoietic Stem Cells/physiology , Humans , Induced Pluripotent Stem Cells/physiology , Karyotyping , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/physiology , Mice , Mice, SCID , Neoplasm Transplantation , Regenerative Medicine/methods , Stem Cell Transplantation/methods , Teratoma/pathology
15.
N Biotechnol ; 28(4): 334-41, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21056705

ABSTRACT

EMSY interacts directly with BRCA2 and links the BRCA2 pathway to sporadic breast and ovarian cancer. It also interacts with BS69 and HP1b, both of which are involved in chromatin remodelling, and with NIF-1 and DBC-1 in the regulation of nuclear receptor-mediated transcription. Here we investigate the function of EMSY during amphibian development, and in doing so provide the first loss-of-function analysis of this protein. Injection of Xenopus tropicalis embryos with antisense morpholino oligonucleotides targeting XtEMSY impairs gastrulation movements, disrupts dorsal structures, and kills embryos by tailbud stages. Consistent with these observations, regional markers such as Xbra, Chd, Gsc, Shh, Sox3 and Sox17 are downregulated. In contrast to these regional markers, expression of p53 is upregulated in such embryos, and at later stages Bax expression is elevated and apoptotic cells can be detected. Our results demonstrate that EMSY has an essential role in development and they provide an in vivo loss-of-function model that might be used to explore the biochemical functions of this protein in more detail.


Subject(s)
Carrier Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation/physiology , Xenopus Proteins/metabolism , Xenopus/embryology , Xenopus/physiology , Animals , Gene Silencing , Transcription Factors
16.
Int J Dev Biol ; 53(1): 37-43, 2009.
Article in English | MEDLINE | ID: mdl-19123125

ABSTRACT

Members of the REEP (Receptor expression enhancing protein) family contain a TB2/DP1, HVA22 domain that is involved in intracellular trafficking and secretion. Consistent with the presence of this domain, REEP1 and REEP3 enhance the expression of odorant and taste receptors in mammals, while mutation of these genes causes defects in neural development. REEP4 was identified in the course of a functional antisense morpholino oligonucleotide screen searching for genes involved in the early development of Xenopus tropicalis: although over-expression of the gene causes no phenotype, embryos lacking REEP4 develop a slightly kinked body axis and are paralysed. At tailbud stages of development, REEP4 is expressed in the somites and neural tube. The paralysis observed in embryos lacking REEP4 might therefore be caused by defects in the nervous system or in muscle. To address this point, we examined the expression of various neural and muscle markers and found that although all are expressed normally at early stages of development, many are down regulated by the tailbud stage. This suggests that REEP4 plays a role in the maintenance of both the nervous system and the musculature.


Subject(s)
Amphibian Proteins/metabolism , Membrane Transport Proteins/metabolism , Paralysis/embryology , Paralysis/metabolism , Xenopus/abnormalities , Xenopus/metabolism , Amino Acid Sequence , Amphibian Proteins/chemistry , Amphibian Proteins/genetics , Animals , Base Sequence , Biomarkers , Conserved Sequence , Down-Regulation , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Molecular Sequence Data , Muscle Development , Paralysis/genetics , Phylogeny , Sequence Alignment , Xenopus/genetics
17.
Dev Dyn ; 237(6): 1718-25, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18498100

ABSTRACT

Kazrin is a recently described desmosomal protein that binds the cornified envelope precursor periplakin. In this study, we have examined kazrin isoform A expression during the development of Xenopus tropicalis and investigated the consequences of its depletion. Whole mount in situ hybridisation revealed that kazrinA mRNA is expressed throughout the embryo at least until tadpole stages. Xenopus tropicalis embryos that had been injected with antisense morpholino oligonucleotides directed against kazrinA failed to elongate properly and showed defects in development of the head, eye, notochord, and somites. We also observed that the epidermis became disorganised and frequently separated from the underlying mesoderm, causing the formation of epidermal blisters. Together, our results suggest that loss of kazrinA causes defects in cell adhesion that affect axial elongation, cell differentiation, and epidermal morphogenesis.


Subject(s)
Carrier Proteins/physiology , Epidermis/embryology , Gene Expression Regulation, Developmental , Xenopus/embryology , Xenopus/metabolism , Animals , Base Sequence , Body Patterning , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Adhesion , Cell Differentiation , Cytoskeleton/metabolism , In Situ Hybridization , Models, Biological , Molecular Sequence Data , Phenotype , RNA, Messenger/metabolism
18.
Traffic ; 8(7): 893-903, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17587408

ABSTRACT

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development.


Subject(s)
Clathrin/metabolism , Protein Serine-Threonine Kinases/physiology , SNARE Proteins/metabolism , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Biological Transport , Cell Membrane/metabolism , Endosomes/metabolism , Evolution, Molecular , HeLa Cells , Humans , Mice , Qa-SNARE Proteins/metabolism , RNA, Small Interfering/metabolism , Xenopus/metabolism
19.
PLoS Genet ; 2(11): e193, 2006 Nov 17.
Article in English | MEDLINE | ID: mdl-17112317

ABSTRACT

To identify novel genes involved in early development, and as proof-of-principle of a large-scale reverse genetics approach in a vertebrate embryo, we have carried out an antisense morpholino oligonucleotide (MO) screen in Xenopus tropicalis, in the course of which we have targeted 202 genes expressed during gastrula stages. MOs were designed to complement sequence between -80 and +25 bases of the initiating AUG codons of the target mRNAs, and the specificities of many were tested by (i) designing different non-overlapping MOs directed against the same mRNA, (ii) injecting MOs differing in five bases, and (iii) performing "rescue" experiments. About 65% of the MOs caused X. tropicalis embryos to develop abnormally (59% of those targeted against novel genes), and we have divided the genes into "synphenotype groups," members of which cause similar loss-of-function phenotypes and that may function in the same developmental pathways. Analysis of the expression patterns of the 202 genes indicates that members of a synphenotype group are not necessarily members of the same synexpression group. This screen provides new insights into early vertebrate development and paves the way for a more comprehensive MO-based analysis of gene function in X. tropicalis.


Subject(s)
Oligonucleotides, Antisense/pharmacology , Xenopus/embryology , Xenopus/genetics , Animals , Apoptosis/drug effects , Body Patterning/drug effects , Databases, Genetic , Embryo, Nonmammalian/abnormalities , Embryo, Nonmammalian/drug effects , Gastrula/drug effects , Gene Expression Regulation, Developmental/drug effects , Mice , Oligonucleotides, Antisense/genetics , Phenotype , Zebrafish/embryology
20.
Dev Biol ; 300(2): 554-69, 2006 Dec 15.
Article in English | MEDLINE | ID: mdl-17055478

ABSTRACT

Recent experiments suggest that Xenopus Neurotrophin Receptor Homolog 1 (NRH1) proteins act through the planar cell polarity pathway to regulate convergent extension movements during gastrulation and neurulation. We show in this paper that NRH1 proteins are also required for the proper expression of mesodermally expressed genes such as Xbra and Chordin, and to a lesser extent, of Xwnt11. Loss of NRH1 function is followed, during gastrula and neurula stages, by a dramatic increase in apoptosis. Apoptosis is delayed by injection of Xbra RNA, suggesting that cell death is a consequence, at least in part, of the down-regulation of this gene, and it is also delayed by expression of activated forms of Rho, Rac and Cdc42. These small GTPases have previously been implicated in the planar cell polarity pathway in Xenopus and, in other systems, in the regulation of apoptosis. We conclude that the effects of NRH1 proteins include the regulation of mesodermal gene expression and that the disruption of gastrulation that is caused by their loss of function is a consequence of the down-regulation of Xbra and other genes, in addition to direct interference with the planar cell polarity pathway. The apoptosis observed in embryos lacking NRH1 function is not an indirect consequence of the disruption of gastrulation, and indeed it may contribute to the observed morphological defects.


Subject(s)
Apoptosis/physiology , Embryo, Nonmammalian/physiology , Gastrula/physiology , Mesoderm/physiology , Receptors, Nerve Growth Factor/physiology , Xenopus Proteins/physiology , Xenopus laevis/embryology , Animals , Base Sequence , Glycoproteins/physiology , Intercellular Signaling Peptides and Proteins/physiology , Molecular Sequence Data , Oligonucleotides, Antisense , Receptors, Nerve Growth Factor/antagonists & inhibitors , Receptors, Nerve Growth Factor/biosynthesis , Receptors, Nerve Growth Factor/genetics , T-Box Domain Proteins/physiology , Wnt Proteins/physiology , Xenopus Proteins/genetics , rho GTP-Binding Proteins/biosynthesis , rho GTP-Binding Proteins/genetics
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