Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory latent TGFß-binding site.

Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFß-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin αvß3-binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease.

Structural studies of elastic fibre and microfibrillar proteins.

Elastic tissues owe their functional properties to the composition of their extracellular matrices, particularly the range of extracellular, multidomain extensible elastic fibre and microfibrillar proteins. These proteins include elastin, fibrillin, latent TGFß binding proteins (LTBPs) and collagens, where their biophysical and biochemical properties not only give the matrix structural integrity, but also play a vital role in the mechanisms that underlie tissue homeostasis. Thus far structural information regarding the structure and hierarchical assembly of these molecules has been challenging and the resolution has been limited due to post-translational modification and their multidomain nature leading to flexibility, which together result in conformational and structural heterogeneity. In this review, we describe some of the matrix proteins found in elastic fibres and the new emerging techniques that can shed light on their structure and dynamic properties.

A new MMP-mediated prodomain cleavage mechanism to activate bone morphogenetic proteins from the extracellular matrix.

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-ßs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

The role of fibrillin and microfibril binding proteins in elastin and elastic fibre assembly.

Fibrillin is a large evolutionarily ancient extracellular glycoprotein that assembles to form beaded microfibrils which are essential components of most extracellular matrices. Fibrillin microfibrils have specific biomechanical properties to endow animal tissues with limited elasticity, a fundamental feature of the durable function of large blood vessels, skin and lungs. They also form a template for elastin deposition and provide a platform for microfibril-elastin binding proteins to interact in elastic fibre assembly. In addition to their structural role, fibrillin microfibrils mediate cell signalling via integrin and syndecan receptors, and microfibrils sequester transforming growth factor (TGF)ß family growth factors within the matrix to provide a tissue store which is critical for homeostasis and remodelling.

Internal cleavage and synergy with twisted gastrulation enhance BMP inhibition by BMPER.

Bone morphogenetic proteins (BMPs) are essential signalling molecules involved in developmental and pathological processes and are regulated in the matrix by secreted glycoproteins. One such regulator is BMP-binding endothelial cell precursor-derived regulator (BMPER) which can both inhibit and enhance BMP signalling in a context and concentration-dependent manner. Twisted gastrulation (Tsg) can also promote or ablate BMP activity but it is unclear whether Tsg and BMPER directly interact and thereby exert a synergistic function on BMP signalling. Here, we show that human BMPER binds to Tsg through the N-terminal BMP-binding region which alone more potently inhibits BMP-4 signalling than full-length BMPER. Additionally, BMPER and Tsg cooperatively inhibit BMP-4 signalling suggesting a synergistic function to dampen BMP activity. Furthermore, full-length BMPER is targeted to the plasma membrane via binding of its C-terminal region to cell surface heparan sulphate proteoglycans but the active cleavage fragment is diffusible. Small-angle X-ray scattering and electron microscopy show that BMPER has an elongated conformation allowing the N-terminal BMP-binding and C-terminal cell-interactive regions to be spatially separated. To gain insight into the regulation of BMPER bioavailability by internal cleavage, a disease-causing BMPER point mutation, P370L, previously identified in the acid-catalysed cleavage site, was introduced. The mutated protein was secreted but the mutation prevented intracellular cleavage resulting in a lack of bioactive cleavage fragment. Furthermore, mutant BMPER was extracellularly cleaved at a downstream site presumably becoming available due to the mutation. This susceptibility to extracellular proteases and loss of bioactive N-terminal cleavage fragment may result in loss of BMPER function in disease.

Multiscale Imaging Reveals the Hierarchical Organization of Fibrillin Microfibrils.

Fibrillin microfibrils are evolutionarily ancient, structurally complex extracellular polymers found in mammalian elastic tissues where they endow elastic properties, sequester growth factors and mediate cell signalling; thus, knowledge of their structure and organization is essential for a more complete understanding of cell function and tissue morphogenesis. By combining multiple imaging techniques, we visualize three levels of hierarchical organization of fibrillin structure ranging from micro-scale fiber bundles in the ciliary zonule to nano-scale individual microfibrils. Serial block-face scanning electron microscopy imaging suggests that bundles of zonule fibers are bound together by circumferential wrapping fibers, which is mirrored on a shorter-length scale where individual zonule fibers are interwoven by smaller fibers. Electron tomography shows that microfibril directionality varies from highly aligned and parallel, connecting to the basement membrane, to a meshwork at the zonule fiber periphery, and microfibrils within the zonule are connected by short cross-bridges, potentially formed by fibrillin-binding proteins. Three-dimensional reconstructions of negative-stain electron microscopy images of purified microfibrils confirm that fibrillin microfibrils have hollow tubular structures with defined bead and interbead regions, similar to tissue microfibrils imaged in our tomograms. These microfibrils are highly symmetrical, with an outer ring and interwoven core in the bead and four linear prongs, each accommodating a fibrillin dimer, in the interbead region. Together these data show how a single molecular building block is organized into different levels of hierarchy from microfibrils to tissue structures spanning nano- to macro-length scales. Furthermore, the application of these combined imaging approaches has wide applicability to other tissue systems.

ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-ß superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill-Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

Defining the hierarchical organisation of collagen VI microfibrils at nanometre to micrometre length scales.

Extracellular matrix microfibrils are critical components of connective tissues with a wide range of mechanical and cellular signalling functions. Collagen VI is a heteromeric network-forming collagen which is expressed in tissues such as skin, lung, blood vessels and articular cartilage where it anchors cells into the matrix allowing for transduction of biochemical and mechanical signals. It is not understood how collagen VI is arranged into microfibrils or how these microfibrils are arranged into tissues. Therefore we have characterised the hierarchical organisation of collagen VI across multiple length scales. The frozen hydrated nanostructure of purified collagen VI microfibrils was reconstructed using cryo-TEM. The bead region has a compact hollow head and flexible tail regions linked by the collagenous interbead region. Serial block face SEM imaging coupled with electron tomography of the pericellular matrix (PCM) of murine articular cartilage revealed that the PCM has a meshwork-like organisation formed from globular densities ∼30nm in diameter. These approaches can characterise structures spanning nanometer to millimeter length scales to define the nanostructure of individual collagen VI microfibrils and the micro-structural organisation of these fibrils within tissues to help in the future design of better mimetics for tissue engineering. STATEMENT OF SIGNIFICANCE: Cartilage is a connective tissue rich in extracellular matrix molecules and is tough and compressive to cushion the bones of joints. However, in adults cartilage is poorly repaired after injury and so this is an important target for tissue engineering. Many connective tissues contain collagen VI, which forms microfibrils and networks but we understand very little about these assemblies or the tissue structures they form. Therefore, we have use complementary imaging techniques to image collagen VI microfibrils from the nano-scale to the micro-scale in order to understand the structure and the assemblies it forms. These findings will help to inform the future design of scaffolds to mimic connective tissues in regenerative medicine applications.

Dysregulated expression of sterol O-acyltransferase 1 (Soat1) in the hair shaft of Hoxc13 null mice.

The cholesterol-metabolizing enzyme sterol O-acetyltransferase (SOAT1) is implicated in an increasing number of biological and pathological processes in a number of organ systems, including the differentiation of the hair shaft. While the functional and regulatory mechanisms underlying these diverse functional roles remain poorly understood, the compartment of the hair shaft known as medulla, affected by mutations in Soat1, may serve as a suitable model for defining some of these mechanisms. A comparative analysis of mRNA and protein expression patterns of Soat1/SOAT1 and the transcriptional regulator Hoxc13/HOXC13 in postnatal skin of FVB/NTac mice indicated co-expression in the most proximal cells of the differentiating medulla. This finding combined with the significant downregulation of Soat1 expression in postnatal skin of both Hoxc13 gene-targeted and transgenic mice based on previously reported DNA microarray results suggests a potential regulatory relationship between the two genes. Non-detectable SOAT1 expression in the defective hair follicle medulla of Hoxc13(tm1Mrc) mice and evidence for binding of HOXC13 to the Soat1 upstream control region obtained by ChIP assay suggests that Soat1 is a downstream regulatory target for HOXC13 during medulla differentiation.

Kiss your tail goodbye: the role of PARN, Nocturnin, and Angel deadenylases in mRNA biology.

PARN, Nocturnin and Angel are three of the multiple deadenylases that have been described in eukaryotic cells. While each of these enzymes appear to target poly(A) tails for shortening and influence RNA gene expression levels and quality control, the enzymes differ in terms of enzymatic mechanisms, regulation and biological impact. The goal of this review is to provide an in depth biochemical and biological perspective of the PARN, Nocturnin and Angel deadenylases. Understanding the shared and unique roles of these enzymes in cell biology will provide important insights into numerous aspects of the post-transcriptional control of gene expression. This article is part of a Special Issue entitled: RNA Decay mechanisms.

In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart, retina, spinal cord, optic nerve, sensory hair cells, and fins. The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 °C, this process occurs within six hours post-amputation (hpa, Figure 1B). Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D). Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E). Depending on the level of the amputation, full regeneration is completed in a week to a month. The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration. Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue. This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

The nude mutant gene Foxn1 is a HOXC13 regulatory target during hair follicle and nail differentiation.

Among the Hox genes, homeobox C13 (Hoxc13) has been shown to be essential for proper hair shaft differentiation, as Hoxc13 gene-targeted (Hoxc13(tm1Mrc)) mice completely lack external hair. Because of the remarkable overt phenotypic parallels to the Foxn1(nu) (nude) mutant mice, we sought to determine whether Hoxc13 and forkhead box N1 (Foxn1) might act in a common pathway of hair follicle (HF) differentiation. We show that the alopecia exhibited by both the Hoxc13(tm1Mrc) and Foxn1(nu) mice is because of strikingly similar defects in hair shaft differentiation and that both mutants suffer from a severe nail dystrophy. These phenotypic similarities are consistent with the extensive overlap between Hoxc13 and Foxn1 expression patterns in the HF and the nail matrix. Furthermore, DNA microarray analysis of skin from Hoxc13(tm1Mrc) mice identified Foxn1 as significantly downregulated along with numerous hair keratin genes. This Foxn1 downregulation apparently reflects the loss of direct transcriptional control by HOXC13 as indicated by our results obtained through co-transfection and chromatin immunoprecipitation (ChIP) assays. As presented in the discussion, these data support a regulatory model of keratinocyte differentiation in which HOXC13-dependent activation of Foxn1 is part of a regulatory cascade controlling the expression of terminal differentiation markers.

Both Hoxc13 orthologs are functionally important for zebrafish tail fin regeneration.

Hox genes are re-expressed during regeneration in many species. Given their important role in body plan development, it has been assumed, but not directly shown, that they play a functional role in regeneration. In this paper we show that morpholino-mediated knockdown of either Hoxc13a or Hoxc13b during the process of zebrafish tail fin regeneration results in a significant reduction of regenerative outgrowth. Furthermore, cellular proliferation within the blastema is directly affected in both knockdowns. Hence, similar to the demonstration of unique functions of multiple Hox genes during limb formation, both Hoxc13 orthologs have distinct functions in regeneration.

A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor.

In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Cre-mediated site-specific recombination in zebrafish embryos.

Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish.

Matrix metalloproteinase expression and function during fin regeneration in zebrafish: analysis of MT1-MMP, MMP2 and TIMP2.

Matrix metalloproteinases (MMPs) play key roles in the turnover of extracellular matrix (ECM) and, thereby, function as key regulators of cell-ECM interactions during development. In spite of their importance during developmental processes, relatively little has been reported about the role of these metalloproteinases during limb development and regeneration. To approach the problem of cell-ECM interactions during limb (fin) regeneration, we have utilized zebrafish as an experimental model. Based on previous MMP cloning studies from our laboratory, the current study has focused on the expression of membrane-type 1 metalloproteinase (MT1-MMP), gelatinase A (MMP-2) and endogenous tissue inhibitor 2 of metalloproteinases (TIMP-2) during fin regeneration in adult zebrafish. In situ analysis indicated co-expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts in regenerating caudal fins. In situ gelatin-zymography confirmed the presence of active metalloproteinases in regenerating fins. zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts were expressed in the blastema and basal epithelium during caudal fin regeneration while expression of type IV collagen [zcol-IV(a5)] transcripts (a basal lamina component) was restricted to the basal epithelium. Fin outgrowth was greatly reduced in the presence of GM6001 (an inhibitor of MMP activity) indicating the importance of these enzymes during fin regeneration. Previous studies by Itoh (EMBO, 2001) indicated that expression of a vertebrate MT1-MMP construct containing only the hemopexin-transmembrane-cytoplasmic domains (MT1HPX) resulted in blockage of MT1-MMP homophilic complex formation and subsequent inhibition of pro-MMP-2 activation. Interference with homophilic complex formation was attributed to expression of the hemopexin domain at the cell surface. Building upon these earlier findings, the current study found that ectopic expression of MT1HPX in fin regenerates inhibited the regeneration process and resulted in a reduction in cell proliferation in the blastema. Taken together, these results indicate that MMPs have an important role during fin regeneration in zebrafish.

Integration of embryonic stem cells in metanephric kidney organ culture.

Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.