ABSTRACT
Proneural transcription factors establish molecular cascades to orchestrate neuronal diversity. One such transcription factor, Atonal homolog 1 (Atoh1), gives rise to cerebellar excitatory neurons and over 30 distinct nuclei in the brainstem critical for hearing, breathing, and balance. Although Atoh1 lineage neurons have been qualitatively described, the transcriptional programs that drive their fate decisions and the full extent of their diversity remain unknown. Here, we analyzed single-cell RNA sequencing and ATOH1 DNA binding in Atoh1 lineage neurons of the developing mouse hindbrain. This high-resolution dataset identified markers for specific brainstem nuclei and demonstrated that transcriptionally heterogeneous progenitors require ATOH1 for proper migration. Moreover, we identified a sizable population of proliferating unipolar brush cell progenitors in the mouse Atoh1 lineage, previously described in humans as the origin of one medulloblastoma subtype. Collectively, our data provide insights into the developing mouse hindbrain and markers for functional assessment of understudied neuronal populations.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Cell Lineage , Neurons , Rhombencephalon , Single-Cell Analysis , Transcriptome , Animals , Rhombencephalon/metabolism , Rhombencephalon/cytology , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Mice , Neurons/metabolism , Neurons/cytology , Cell Lineage/genetics , Single-Cell Analysis/methods , Transcriptome/genetics , Cell Differentiation , Gene Expression Regulation, Developmental , Neurogenesis/genetics , Cell MovementABSTRACT
Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Female , Male , Mice , Animals , Gene Editing/methods , RNA, Guide, CRISPR-Cas Systems , Mutation , Zygote/metabolism , Animals, Genetically Modified , OocytesABSTRACT
Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.
ABSTRACT
Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood-brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.
Subject(s)
Blood-Brain Barrier , Capsid , Mice , Animals , Blood-Brain Barrier/metabolism , Capsid/metabolism , Genetic Vectors , Central Nervous System/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , Dependovirus/genetics , Dependovirus/metabolismABSTRACT
SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.
Subject(s)
Heart Defects, Congenital , Heart Septal Defects, Ventricular , Animals , Mice , Endocardium/metabolism , Heart , Heart Defects, Congenital/genetics , Heart Septal Defects, Ventricular/genetics , Heart Septal Defects, Ventricular/metabolism , Myocardium/metabolism , SOXF Transcription Factors/metabolismABSTRACT
Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but pituitary hormone disorders are not fully understood. Herein we report that genetically-engineered mice with deletion of the hedgehog signaling receptor Patched1 by S100a4 promoter-driven Cre recombinase (S100a4-Cre;Ptch1fl/fl mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adult, S100a4-Cre;Ptch1fl/fl mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45+ cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.
Subject(s)
Hypogonadism/metabolism , Integrases/metabolism , Patched-1 Receptor/metabolism , Pituitary Gland/metabolism , S100 Calcium-Binding Protein A4/metabolism , Animals , Epididymis/pathology , Female , Humans , Hypogonadism/genetics , Hypogonadism/pathology , Male , Mice , Mice, Knockout , Ovary/pathology , Patched-1 Receptor/genetics , Pituitary Gland, Anterior/metabolism , Reproduction/physiology , Seminal Vesicles/pathology , Sexual Maturation , Signal Transduction , Testis , Testosterone/blood , Uterus/pathologyABSTRACT
Ovarian theca androgen production is regulated by the pituitary LH and intrafollicular factors. Enhanced androgen biosynthesis by theca cells contributes to polycystic ovary syndrome (PCOS) in women, but the ovarian consequences of elevated androgens are not completely understood. Our study documents the molecular events that are altered in the theca and stromal cells of mice exposed to high androgen levels, using the nonaromatizable androgen DHT. Changes in ovarian morphology and function were observed not only in follicles, but also in the stromal compartment. Genome-wide microarray analyses revealed marked changes in the ovarian transcriptome of DHT-treated females within 1 week. Particularly striking was the increased expression of vascular cell adhesion molecule 1 (Vcam1) specifically in the NR2F2/COUPTF-II lineage theca cells, not granulosa cells, of growing follicles and throughout the stroma of the androgen-treated mice. This response was mediated by androgen receptors (ARs) present in theca and stromal cells. Human theca-derived cultures expressed both ARs and NR2F2 that were nuclear. VCAM1 mRNA and protein were higher in PCOS-derived theca cells compared with control theca and reduced markedly by the AR antagonist flutamide. In the DHT-treated mice, VCAM1 was transiently induced by equine chorionic gonadotropin, when androgen and estrogen biosynthesis peak in preovulatory follicles, and was potently suppressed by a superovulatory dose of human chorionic gonadotropin. High levels of VCAM1 in the theca and interstitial cells of DHT-treated mice and in adult Leydig cells indicate that there may be novel functions for VCAM1 in reproductive tissues, including the gonads.
Subject(s)
Dihydrotestosterone , Hyperandrogenism/metabolism , Ovarian Follicle/metabolism , Ovary/metabolism , Stromal Cells/metabolism , Theca Cells/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Animals , COUP Transcription Factor II/metabolism , Female , Hyperandrogenism/chemically induced , Mice , Receptors, Androgen/metabolismABSTRACT
This data is a curated collection of visual images of gene expression patterns from the pre- and post-natal mouse lung, accompanied by associated mRNA probe sequences and RNA-Seq expression profiles. Mammalian lungs undergo significant growth and cellular differentiation before and after the transition to breathing air. Documenting normal lung development is an important step in understanding abnormal lung development, as well as the challenges faced during a preterm birth. Images in this dataset indicate the spatial distribution of mRNA transcripts for over 500 different genes that are active during lung development, as initially determined via RNA-Seq. Images were systematically acquired using high-throughput in situ hybridization with non-radioactive digoxigenin-labeled mRNA probes across mouse lungs from developmental time points E16.5, E18.5, P7, and P28. The dataset was produced as part of The Molecular Atlas of Lung Development Program (LungMAP) and is hosted at https://lungmap.net. This manuscript describes the nature of the data and the protocols for generating the dataset.
ABSTRACT
We compute the x-ray emission spectrum of liquid methanol, with the dynamical effects that result from the creation of the core hole included in a semiclassical way. Our method closely reproduces a fully quantum mechanical description of the dynamical effects for relevant one-dimensional models of the hydrogen-bonded methanol molecules. For the liquid, we find excellent agreement with the experimental spectrum, including the large isotope effect in the first split peak. The dynamical effects depend sensitively on the initial structure in terms of the local hydrogen-bonding (H-bonding) character: non-donor molecules contribute mainly to the high-energy peak while molecules with a strong donating H-bond contribute to the peak at lower energy. The spectrum thus reflects the initial structure mediated by the dynamical effects that are, however, seen to be crucial in order to reproduce the intensity distribution of the recently measured spectrum.
ABSTRACT
Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
Subject(s)
Abnormalities, Multiple/genetics , Endopeptidases/genetics , Intellectual Disability/genetics , Adolescent , Animals , Child , Child, Preschool , Disease Models, Animal , Female , Gene Deletion , Humans , Male , Mice , Pedigree , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Seizures/geneticsABSTRACT
Gain-of-function mutations in some genes underlie neurodegenerative conditions, whereas loss-of-function mutations in the same genes have distinct phenotypes. This appears to be the case with the protein ataxin 1 (ATXN1), which forms a transcriptional repressor complex with capicua (CIC). Gain of function of the complex leads to neurodegeneration, but ATXN1-CIC is also essential for survival. We set out to understand the functions of the ATXN1-CIC complex in the developing forebrain and found that losing this complex results in hyperactivity, impaired learning and memory, and abnormal maturation and maintenance of upper-layer cortical neurons. We also found that CIC activity in the hypothalamus and medial amygdala modulates social interactions. Informed by these neurobehavioral features in mouse mutants, we identified five individuals with de novo heterozygous truncating mutations in CIC who share similar clinical features, including intellectual disability, attention deficit/hyperactivity disorder (ADHD), and autism spectrum disorder. Our study demonstrates that loss of ATXN1-CIC complexes causes a spectrum of neurobehavioral phenotypes.
Subject(s)
Ataxin-1/genetics , Autism Spectrum Disorder/genetics , Neurodegenerative Diseases/genetics , Nuclear Proteins/genetics , Repressor Proteins/genetics , Animals , Cerebellum/pathology , Female , Humans , Intellectual Disability/genetics , Interpersonal Relations , Male , Mice , Nerve Tissue Proteins/genetics , PhenotypeABSTRACT
BACKGROUND: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated. RESULTS: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion. CONCLUSIONS: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.
Subject(s)
Disease Models, Animal , Focal Dermal Hypoplasia/genetics , Gene Deletion , Membrane Proteins/genetics , Mice , Acyltransferases , Animals , Breeding , Cell Line , Chimera , Focal Dermal Hypoplasia/metabolism , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Knockout Techniques , Gene Silencing , Gene Targeting , Genes, Lethal , Humans , Introns , Membrane Proteins/metabolism , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Wnt3A Protein/metabolism , Zygote/metabolismABSTRACT
We report x-ray emission and absorption spectroscopy studies of the electronic structure of the predissociative α(3) phase of CO bound at hollow sites of Fe(100) as well as of the on-top bound species in the high-coverage α(1) phase. The analysis is supported by density functional calculations of structures and spectra. The bonding of "lying down" CO in the hollow site is well described in terms of π to π∗ charge transfer made possible through bonding interaction also at the oxygen in the minority spin-channel. The on-top CO in the mixed, high-coverage α(1) phase is found to be tilted due to adsorbate-adsorbate interaction, but still with bonding mainly characteristic of "vertical" on-top adsorbed CO similar to other transition-metal surfaces.
Subject(s)
Carbon Monoxide/chemistry , Iron/chemistry , Quantum Theory , Spectrometry, X-Ray EmissionABSTRACT
Tauopathies, characterized by neurofibrillary tangles (NFTs) of phosphorylated tau proteins, are a group of neurodegenerative diseases, including frontotemporal dementia and both sporadic and familial Alzheimer's disease. Forebrain-specific over-expression of human tau(P301L), a mutation associated with frontotemporal dementia with parkinsonism linked to chromosome 17, in rTg4510 mice results in the formation of NFTs, learning and memory impairment and massive neuronal death. Here, we show that the mRNA and protein levels of NMNAT2 (nicotinamide mononucleotide adenylyltransferase 2), a recently identified survival factor for maintaining neuronal health in peripheral nerves, are reduced in rTg4510 mice prior to the onset of neurodegeneration or cognitive deficits. Two functional cAMP-response elements (CREs) were identified in the nmnat2 promoter region. Both the total amount of phospho-CRE binding protein (CREB) and the pCREB bound to nmnat2 CRE sites in the cortex and the hippocampus of rTg4510 mice are significantly reduced, suggesting that NMNAT2 is a direct target of CREB under physiological conditions and that tau(P301L) overexpression down-regulates CREB-mediated transcription. We found that over-expressing NMNAT2 or its homolog NMNAT1, but not NMNAT3, in rTg4510 hippocampi from 6 weeks of age using recombinant adeno-associated viral vectors significantly reduced neurodegeneration caused by tau(P301L) over-expression at 5 months of age. In summary, our studies strongly support a protective role of NMNAT2 in the mammalian central nervous system. Decreased endogenous NMNAT2 function caused by reduced CREB signaling during pathological insults may be one of underlying mechanisms for neuronal death in tauopathies.
Subject(s)
CREB-Binding Protein/genetics , Down-Regulation , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Tauopathies/genetics , Transcription, Genetic , Animals , Base Sequence , Blotting, Western , DNA Primers , Disease Models, Animal , Fluorescent Antibody Technique , Hippocampus/pathology , Humans , Mice , Mice, Transgenic , Promoter Regions, Genetic , Real-Time Polymerase Chain Reaction , Tauopathies/pathologyABSTRACT
Radioimmunotherapy involves the use of radiolabeled monoclonal antibodies (MAbs) to treat malignancy. The therapeutic effect is determined by the radiopharmaceutical, the radiation absorbed dose and previous treatments. There are currently two approved radiopharmaceuticals for the treatment of B-cell lymphoma - the (90)Y-labeled ibritumomab and the (131)I-labeled tositumomab. Both are directed against CD20, albeit not against the same epitope. This paper summarizes current results of dose-responses for normal tissues and tumours of [(131)I]tositumomab and [(90)Y]ibritumomab tiuxetan, discusses them in the context of dosimetry methods used and highlights the assumptions being made in the different dosimetry methodologies. Moreover, we wish to point at the possibility of performing low-cost therapy bremsstrahlung imaging for [(90)Y]ibritumomab tiuxetan to confirm biodistribution, and possibly also for dosimetric calculations.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/therapeutic use , Iodine Radioisotopes/administration & dosage , Iodine Radioisotopes/therapeutic use , Lymphoma, B-Cell/radiotherapy , Radioimmunotherapy/methods , Yttrium Radioisotopes/administration & dosage , Yttrium Radioisotopes/therapeutic use , Antibodies, Monoclonal/pharmacokinetics , Antigens, CD20/metabolism , Bone Marrow/radiation effects , Dose-Response Relationship, Radiation , Female , Humans , Lymphoma, B-Cell/diagnostic imaging , Lymphoma, B-Cell/immunology , Male , Radiopharmaceuticals/administration & dosage , Radiopharmaceuticals/pharmacokinetics , Radiopharmaceuticals/therapeutic use , Radiotherapy Planning, Computer-Assisted , Tomography, Emission-Computed, Single-Photon , Tomography, X-Ray Computed , Yttrium Radioisotopes/pharmacokineticsABSTRACT
We apply the Kramers-Heisenberg formula to a model water dimer to discuss vibrational interference in the x-ray emission spectrum of the donor molecule for which the core-ionized potential energy surface is dissociative but bounded by the accepting molecule. A long core-hole lifetime leads to decay from Zundel-like, fully delocalized vibrational states in the intermediate potential without involvement of a specific dissociated component. Comparison is made to a model with an unbound intermediate state allowing dissociation to infinity which gives a sharp, fully dissociated feature, and a broad molecular peak at long core-hole life time. The implications of the vibrational interference effect on the liquid water spectrum are discussed and it is proposed that this mainly gives rise to an isotope-dependent asymmetrical broadening of the lone pair peak.
ABSTRACT
In this study, we simulated a Siemens E.CAM SPECT system using SIMIND Monte Carlo program to acquire its experimental characterization in terms of energy resolution, sensitivity, spatial resolution and imaging of phantoms using (99m)Tc. The experimental and simulation data for SPECT imaging was acquired from a point source and Jaszczak phantom. Verification of the simulation was done by comparing two sets of images and related data obtained from the actual and simulated systems. Image quality was assessed by comparing image contrast and resolution. Simulated and measured energy spectra (with or without a collimator) and spatial resolution from point sources in air were compared. The resulted energy spectra present similar peaks for the gamma energy of (99m)Tc at 140 KeV. FWHM for the simulation calculated to 14.01 KeV and 13.80 KeV for experimental data, corresponding to energy resolution of 10.01 and 9.86% compared to defined 9.9% for both systems, respectively. Sensitivities of the real and virtual gamma cameras were calculated to 85.11 and 85.39 cps/MBq, respectively. The energy spectra of both simulated and real gamma cameras were matched. Images obtained from Jaszczak phantom, experimentally and by simulation, showed similarity in contrast and resolution. SIMIND Monte Carlo could successfully simulate the Siemens E.CAM gamma camera. The results validate the use of the simulated system for further investigation, including modification, planning, and developing a SPECT system to improve the quality of images.
ABSTRACT
Electronic structure calculations have become an indispensable tool in many areas of materials science and quantum chemistry. Even though the Kohn-Sham formulation of the density-functional theory (DFT) simplifies the many-body problem significantly, one is still confronted with several numerical challenges. In this article we present the projector augmented-wave (PAW) method as implemented in the GPAW program package (https://wiki.fysik.dtu.dk/gpaw) using a uniform real-space grid representation of the electronic wavefunctions. Compared to more traditional plane wave or localized basis set approaches, real-space grids offer several advantages, most notably good computational scalability and systematic convergence properties. However, as a unique feature GPAW also facilitates a localized atomic-orbital basis set in addition to the grid. The efficient atomic basis set is complementary to the more accurate grid, and the possibility to seamlessly switch between the two representations provides great flexibility. While DFT allows one to study ground state properties, time-dependent density-functional theory (TDDFT) provides access to the excited states. We have implemented the two common formulations of TDDFT, namely the linear-response and the time propagation schemes. Electron transport calculations under finite-bias conditions can be performed with GPAW using non-equilibrium Green functions and the localized basis set. In addition to the basic features of the real-space PAW method, we also describe the implementation of selected exchange-correlation functionals, parallelization schemes, ΔSCF-method, x-ray absorption spectra, and maximally localized Wannier orbitals.