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In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts., (© 2024. The Author(s).)

There is a need to define regions of gene activation or repression that control human kidney cells in states of health, injury, and repair to understand the molecular pathogenesis of kidney disease and design therapeutic strategies. Comprehensive integration of gene expression with epigenetic features that define regulatory elements remains a significant challenge. We measure dual single nucleus RNA expression and chromatin accessibility, DNA methylation, and H3K27ac, H3K4me1, H3K4me3, and H3K27me3 histone modifications to decipher the chromatin landscape and gene regulation of the kidney in reference and adaptive injury states. We establish a spatially-anchored epigenomic atlas to define the kidney's active, silent, and regulatory accessible chromatin regions across the genome. Using this atlas, we note distinct control of adaptive injury in different epithelial cell types. A proximal tubule cell transcription factor network of ELF3, KLF6, and KLF10 regulates the transition between health and injury, while in thick ascending limb cells this transition is regulated by NR2F1. Further, combined perturbation of ELF3, KLF6, and KLF10 distinguishes two adaptive proximal tubular cell subtypes, one of which manifested a repair trajectory after knockout. This atlas will serve as a foundation to facilitate targeted cell-specific therapeutics by reprogramming gene regulatory networks., (© 2024. The Author(s).)

Kidney stone disease causes significant morbidity and increases health care utilization. In this work, we decipher the cellular and molecular niche of the human renal papilla in patients with calcium oxalate (CaOx) stone disease and healthy subjects. In addition to identifying cell types important in papillary physiology, we characterize collecting duct cell subtypes and an undifferentiated epithelial cell type that was more prevalent in stone patients. Despite the focal nature of mineral deposition in nephrolithiasis, we uncover a global injury signature characterized by immune activation, oxidative stress and extracellular matrix remodeling. We also identify the association of MMP7 and MMP9 expression with stone disease and mineral deposition, respectively. MMP7 and MMP9 are significantly increased in the urine of patients with CaOx stone disease, and their levels correlate with disease activity. Our results define the spatial molecular landscape and specific pathways contributing to stone-mediated injury in the human papilla and identify associated urinary biomarkers., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods 1 . Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations., (© 2023. The Author(s).)

Kidney Precision Medicine Project (KPMP) is building a spatially specified human kidney tissue atlas in health and disease with single-cell resolution. Here, we describe the construction of an integrated reference map of cells, pathways, and genes using unaffected regions of nephrectomy tissues and undiseased human biopsies from 56 adult subjects. We use single-cell/nucleus transcriptomics, subsegmental laser microdissection transcriptomics and proteomics, near-single-cell proteomics, 3D and CODEX imaging, and spatial metabolomics to hierarchically identify genes, pathways, and cells. Integrated data from these different technologies coherently identify cell types/subtypes within different nephron segments and the interstitium. These profiles describe cell-level functional organization of the kidney following its physiological functions and link cell subtypes to genes, proteins, metabolites, and pathways. They further show that messenger RNA levels along the nephron are congruent with the subsegmental physiological activity. This reference atlas provides a framework for the classification of kidney disease when multiple molecular mechanisms underlie convergent clinical phenotypes.

Comprehensive characterization of cellular heterogeneity and the underlying regulatory landscapes of tissues and organs requires a highly robust and scalable method to acquire matched RNA and chromatin accessibility profiles on the same cells. Here, we describe a single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2) assay, implemented with cellular combinatorial indexing. This method involves tagmentation within permeabilized and fixed single-nucleus isolates to capture accessible chromatin (AC) regions, followed by the capture and reverse transcription of RNA transcripts. Through combinatorial split pool ligations, cDNA and AC within each single nucleus become appended with a common cell barcode combination. The captured cDNA and AC are then co-amplified before splitting and enrichment into single-nucleus RNA and single-nucleus AC sequencing libraries. This protocol is compatible with both nuclei and whole cells and can be completed in 3.5 d. SNARE-seq2 permits robust generation of high-quality, joint single-cell RNA and AC sequencing libraries from hundreds of thousands of single cells per experiment., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals 1 . Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations., (© 2021. The Author(s).)

Comprehensive and spatially mapped molecular atlases of organs at a cellular level are a critical resource to gain insights into pathogenic mechanisms and personalized therapies for diseases. The Kidney Precision Medicine Project (KPMP) is an endeavor to generate three-dimensional (3-D) molecular atlases of healthy and diseased kidney biopsies by using multiple state-of-the-art omics and imaging technologies across several institutions. Obtaining rigorous and reproducible results from disparate methods and at different sites to interrogate biomolecules at a single-cell level or in 3-D space is a significant challenge that can be a futile exercise if not well controlled. We describe a "follow the tissue" pipeline for generating a reliable and authentic single-cell/region 3-D molecular atlas of human adult kidney. Our approach emphasizes quality assurance, quality control, validation, and harmonization across different omics and imaging technologies from sample procurement, processing, storage, shipping to data generation, analysis, and sharing. We established benchmarks for quality control, rigor, reproducibility, and feasibility across multiple technologies through a pilot experiment using common source tissue that was processed and analyzed at different institutions and different technologies. A peer review system was established to critically review quality control measures and the reproducibility of data generated by each technology before their being approved to interrogate clinical biopsy specimens. The process established economizes the use of valuable biopsy tissue for multiomics and imaging analysis with stringent quality control to ensure rigor and reproducibility of results and serves as a model for precision medicine projects across laboratories, institutions and consortia.

The UPF3B-dependent branch of the nonsense-mediated RNA decay (NMD) pathway is critical for human cognition. Here, we examined the role of UPF3B in the olfactory system. Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. UPF3B also regulates the expression of a large cadre of antimicrobial genes in OSNs, and promotes the selection of specific olfactory receptor ( Olfr ) genes for expression in mature OSNs (mOSNs). RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b -null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. Together, our data provides a valuable resource for the olfactory field and insights into the roles of NMD in vivo., Competing Interests: KT, SJ, BL, JD, ES, LZ, SC, AS, SP, RG, KZ, HC, MW No competing interests declared, (© 2020, Tan et al.)

Single-cell RNA sequencing can reveal the transcriptional state of cells, yet provides little insight into the upstream regulatory landscape associated with open or accessible chromatin regions. Joint profiling of accessible chromatin and RNA within the same cells would permit direct matching of transcriptional regulation to its outputs. Here, we describe droplet-based single-nucleus chromatin accessibility and mRNA expression sequencing (SNARE-seq), a method that can link a cell's transcriptome with its accessible chromatin for sequencing at scale. Specifically, accessible sites are captured by Tn5 transposase in permeabilized nuclei to permit, within many droplets in parallel, DNA barcode tagging together with the mRNA molecules from the same cells. To demonstrate the utility of SNARE-seq, we generated joint profiles of 5,081 and 10,309 cells from neonatal and adult mouse cerebral cortices, respectively. We reconstructed the transcriptome and epigenetic landscapes of major and rare cell types, uncovered lineage-specific accessible sites, especially for low-abundance cells, and connected the dynamics of promoter accessibility with transcription level during neurogenesis.

Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Defining cellular and molecular identities within the kidney is necessary to understand its organization and function in health and disease. Here we demonstrate a reproducible method with minimal artifacts for single-nucleus Droplet-based RNA sequencing (snDrop-Seq) that we use to resolve thirty distinct cell populations in human adult kidney. We define molecular transition states along more than ten nephron segments spanning two major kidney regions. We further delineate cell type-specific expression of genes associated with chronic kidney disease, diabetes and hypertension, providing insight into possible targeted therapies. This includes expression of a hypertension-associated mechano-sensory ion channel in mesangial cells, and identification of proximal tubule cell populations defined by pathogenic expression signatures. Our fully optimized, quality-controlled transcriptomic profiling pipeline constitutes a tool for the generation of healthy and diseased molecular atlases applicable to clinical samples.

Scottish terriers (ST) frequently have increased serum alkaline phosphatase (ALP) of the steroid isoform. Many of these also have high serum concentrations of adrenal sex steroids. The study's objective was to determine the cause of increased sex steroids in ST with increased ALP. Adrenal gland suppression and stimulation were compared by low dose dexamethasone (LDDS), human chorionic gonadotropin (HCG) and adrenocorticotropic hormone (ACTH) response tests. Resting plasma pituitary hormones were measured. Steroidogenesis-related mRNA expression was evaluated in six ST with increased ALP, eight dogs of other breeds with pituitary-dependent hyperadrenocorticism (HAC), and seven normal dogs. The genome-wide association of single nucleotide polymorphisms (SNP) with ALP activity was evaluated in 168 ST. ALP (reference interval 8-70 U/L) was high in all ST (1,054 U/L) and HAC (985 U/L) dogs. All HAC dogs and 2/8 ST had increased cortisol post-ACTH administration. All ST and 2/7 Normal dogs had increased sex steroids post-ACTH. ST and Normal dogs had similar post-challenge adrenal steroid profiles following LDDS and HCG. Surprisingly, mRNA of hydroxysteroid 17-beta dehydrogenase 2 (HSD17B2) was lower in ST and Normal dogs than HAC. HSD17B2 facilities metabolism of sex steroids. A SNP region was identified on chromosome 5 in proximity to HSD17B2 that correlated with increased serum ALP. ST in this study with increased ALP had a normal pituitary-adrenal axis in relationship to glucocorticoids and luteinizing hormone. We speculate the identified SNP and HSD17B2 gene may have a role in the pathogenesis of elevated sex steroids and ALP in ST.

A variety of inflammatory conditions of unknown cause (meningoencephalomyelitis of unknown etiology-MUE) and neoplastic diseases can affect the central nervous system (CNS) of dogs. MUE can mimic intracranial neoplasia both clinically, radiologically and even in some cases, histologically. Serum immunosignature protein microarray assays have been used in humans to identify CNS diseases such as Alzheimer's and neoplasia, and in dogs, to detect lymphoma and its progression. This study evaluated the effectiveness of immunosignature profiles for distinguishing between three cohorts of dogs: healthy, intracranial neoplasia, and MUE. Using the learned peptide patterns for these three cohorts, classification prediction was evaluated for the same groups using a 10-fold cross validation methodology. Accuracy for classification was 100%, as well as 100% specific and 100% sensitive. This pilot study demonstrates that immunosignature profiles may help serve as a minimally invasive tool to distinguish between MUE and intracranial neoplasia in dogs.

Detailed characterization of the cell types in the human brain requires scalable experimental approaches to examine multiple aspects of the molecular state of individual cells, as well as computational integration of the data to produce unified cell-state annotations. Here we report improved high-throughput methods for single-nucleus droplet-based sequencing (snDrop-seq) and single-cell transposome hypersensitive site sequencing (scTHS-seq). We used each method to acquire nuclear transcriptomic and DNA accessibility maps for >60,000 single cells from human adult visual cortex, frontal cortex, and cerebellum. Integration of these data revealed regulatory elements and transcription factors that underlie cell-type distinctions, providing a basis for the study of complex processes in the brain, such as genetic programs that coordinate adult remyelination. We also mapped disease-associated risk variants to specific cellular populations, which provided insights into normal and pathogenic cellular processes in the human brain. This integrative multi-omics approach permits more detailed single-cell interrogation of complex organs and tissues.

Significant heterogeneities in gene expression among individual cells are typically interrogated using single whole cell approaches. However, tissues that have highly interconnected processes, such as in the brain, present unique challenges. Single-nucleus RNA sequencing (SNS) has emerged as an alternative method of assessing a cell's transcriptome through the use of isolated nuclei. However, studies directly comparing expression data between nuclei and whole cells are lacking. Here, we have characterized nuclear and whole cell transcriptomes in mouse single neurons and provided a normalization strategy to reduce method-specific differences related to the length of genic regions. We confirmed a high concordance between nuclear and whole cell transcriptomes in the expression of cell type and metabolic modeling markers, but less so for a subset of genes associated with mitochondrial respiration. Therefore, our results indicate that single-nucleus transcriptome sequencing provides an effective means to profile cell type expression dynamics in previously inaccessible tissues.

The developmental origins of most adult stem cells are poorly understood. Here, we report the identification of a transcription factor-RHOX10-critical for the initial establishment of spermatogonial stem cells (SSCs). Conditional loss of the entire 33-gene X-linked homeobox gene cluster that includes Rhox10 causes progressive spermatogenic decline, a phenotype indistinguishable from that caused by loss of only Rhox10. We demonstrate that this phenotype results from dramatically reduced SSC generation. By using a battery of approaches, including single-cell-RNA sequencing (scRNA-seq) analysis, we show that Rhox10 drives SSC generation by promoting pro-spermatogonia differentiation. Rhox10 also regulates batteries of migration genes and promotes the migration of pro-spermatogonia into the SSC niche. The identification of an X-linked homeobox gene that drives the initial generation of SSCs has implications for the evolution of X-linked gene clusters and sheds light on regulatory mechanisms influencing adult stem cell generation in general., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The human brain has enormously complex cellular diversity and connectivities fundamental to our neural functions, yet difficulties in interrogating individual neurons has impeded understanding of the underlying transcriptional landscape. We developed a scalable approach to sequence and quantify RNA molecules in isolated neuronal nuclei from a postmortem brain, generating 3227 sets of single-neuron data from six distinct regions of the cerebral cortex. Using an iterative clustering and classification approach, we identified 16 neuronal subtypes that were further annotated on the basis of known markers and cortical cytoarchitecture. These data demonstrate a robust and scalable method for identifying and categorizing single nuclear transcriptomes, revealing shared genes sufficient to distinguish previously unknown and orthologous neuronal subtypes as well as regional identity and transcriptomic heterogeneity within the human brain., (Copyright © 2016, American Association for the Advancement of Science.)

Epithelial folding is a critical process underlying many morphogenetic events including vertebrate neural tube closure, however, its spatial regulation is largely unknown. Here we show that during neural tube formation Rab11-positive recycling endosomes acquire bilaterally symmetric distribution in the Xenopus neural plate, being enriched at medial apical cell junctions. This mediolateral polarization was under the control of planar cell polarity (PCP) signalling, was necessary for neural plate folding and was accompanied by the polarization of the exocyst component Sec15. Our further experiments demonstrate that similar PCP-dependent polarization of Rab11 is essential for ectopic apical constriction driven by the actin-binding protein Shroom and during embryonic wound repair. We propose that anisotropic membrane trafficking has key roles in diverse morphogenetic behaviours of individual cells and propagates in a tissue by a common mechanism that involves PCP.

With their capability to undergo unlimited self-renewal and to differentiate into all cell types in the body, human embryonic stem cells (hESCs) hold great promise in human cell therapy. However, there are limited tools for easily identifying and isolating live hESC-derived cells. To track hESC-derived neural progenitor cells (NPCs), we applied homologous recombination to knock-in the mCherry gene into the Nestin locus of hESCs. This facilitated the genetic labeling of Nestin positive neural progenitor cells with mCherry. Our reporter system enables the visualization of neural induction from hESCs both in vitro (embryoid bodies) and in vivo (teratomas). This system also permits the identification of different neural subpopulations based on the intensity of our fluorescent reporter. In this context, a high level of mCherry expression showed enrichment for neural progenitors, while lower mCherry corresponded with more committed neural states. Combination of mCherry high expression with cell surface antigen staining enabled further enrichment of hESC-derived NPCs. These mCherry(+) NPCs could be expanded in culture and their differentiation resulted in a down-regulation of mCherry consistent with the loss of Nestin expression. Therefore, we have developed a fluorescent reporter system that can be used to trace neural differentiation events of hESCs.

Background: Xenopus embryonic skin is composed of the superficial layer with defined apicobasal polarity and the inner layer lacking the apical domain. Multiciliated cells (MCCs) originate in the inner layer of the epidermal ectoderm and subsequently migrate to the surface. How MCCs acquire the apicobasal polarity and intercalate into the superficial layer during neurulation is largely unknown. As Rab11-dependent vesicle trafficking has been implicated in ciliary membrane assembly and in apical domain formation in epithelial cells, we assessed the involvement of Rab11 in MCC development., Results: Here we report that Rab11 is specifically enriched and becomes apically polarized in skin MCCs. Interference with Rab11 function by overexpression of a dominant negative mutant or injection of a specific morpholino oligonucleotide inhibited MCC intercalation into the superficial layer. Dominant negative Rab11-expressing MCC precursors revealed intrinsic apicobasal polarity, characterized by the apical domain, which is not normally observed in inner layer cells. Despite the presence of the apical domain, the cells with inhibited Rab11 function were randomly oriented relative to the plane of the tissue, thereby demonstrating a defect in planar polarity., Conclusions: These results establish a requirement for Rab11 in MCC development and support a two-step model, in which the initial polarization of MCC precursors is critical for their integration into the superficial cell layer., (Copyright © 2012 Wiley Periodicals, Inc.)

Reprogramming of the somatic state to pluripotency can be induced by a defined set of transcription factors including Oct3/4, Sox2, Klf4, and c-Myc [Cell 2006;126:663-676]. These induced pluripotent stem cells (iPSCs) hold great promise in human therapy and disease modeling. However, tumor suppressive activities of p53, which are necessary to prevent persistence of DNA damage in mammalian cells, have proven a serious impediment to formation of iPSCs [Nat Methods 2011;8:409-412]. We examined the requirement for downstream p53 activities in suppressing efficiency of reprogramming as well as preventing persistence of DNA damage into the early iPSCs. We discovered that the majority of the p53 activation occurred through early reprogramming-induced DNA damage with the activated expression of the apoptotic inducer Puma and the cell cycle inhibitor p21. While Puma deficiency increases reprogramming efficiency only in the absence of c-Myc, double deficiency of Puma and p21 has achieved a level of efficiency that exceeded that of p53 deficiency alone. We further demonstrated that, in both the presence and absence of p21, Puma deficiency was able to prevent any increase in persistent DNA damage in early iPSCs. This may be due to a compensatory cellular senescent response to reprogramming-induced DNA damage in pre-iPSCs. Therefore, our findings provide a potentially safe approach to enhance iPSC derivation by transiently silencing Puma and p21 without compromising genomic integrity., (Copyright © 2012 AlphaMed Press.)

The planar cell polarity (PCP) pathway organizes the cytoskeleton and polarizes cells within embryonic tissue. We investigate the relationship between PCP signaling and cell fate determination during asymmetric division of neural progenitors (NPs) in mouse embryos. The cortex of Lp/Lp (Loop-tail) mice deficient in the essential PCP mediator Vangl2, homologue of Drosophila melanogaster Strabismus (Stbm), revealed precocious differentiation of neural progenitors into early-born neurons at the expense of late-born neurons and glia. Although Lp/Lp NPs were easily maintained in vitro, they showed premature differentiation and loss of asymmetric distribution of Leu-Gly-Asn-enriched protein (LGN)/partner of inscuteable (Pins), a regulator of mitotic spindle orientation. Furthermore, we observed a decreased frequency in asymmetric distribution of the LGN target nuclear mitotic apparatus protein (NuMa) in Lp/Lp cortical progenitors in vivo. This was accompanied by an increase in the number of vertical cleavage planes typically associated with equal daughter cell identities. These findings suggest that Stbm/Vangl2 functions to maintain cortical progenitors and regulates mitotic spindle orientation during asymmetric divisions in the vertebrate brain.

The development of novel therapeutic strategies for breast cancer requires the identification of molecular targets involved in malignancy. Human Pygopus (Pygo)-1 and -2 are recently discovered components of the Wnt signaling pathway required for beta-Catenin/Tcf dependent transcription in embryos and colorectal cancer cells, but the role of these proteins in malignant cell growth and survival has not yet been determined. We report the expression and requirement for proliferation of hPygo2 in breast cancer cells. hPygo2 protein was overexpressed in malignant breast tumors and in the nuclei of five breast cancer cell lines, but was not expressed in the nuclei of non-malignant breast cells. Phosphorothioated antisense oligonucleotides were used to specifically knockdown expression hPygo2 in Mcf-7 and MDA-MB-468 cell lines. hPygo2 was required for the growth, in tissue culture and anchorage-independent assays, of both cell lines and for the expression of the Wnt target gene Cyclin D1. We conclude that hPygo2 is highly expressed in, and required for the growth of breast carcinoma cells.

Purpose: The Pygopus proteins are critical elements of the canonical Wnt/beta-catenin transcriptional complex. In epithelial ovarian cancer, constitutively active Wnt signaling is restricted to one (endometrioid) tumor subtype. The purpose of this study was to determine the level of expression and growth requirements of human Pygopus2 (hPygo2) protein in epithelial ovarian cancer., Experimental Design: Expression and subcellular localization of hPygo2 was determined in epithelial ovarian cancer cell lines and tumors using Northern blot, immunoblot, and immunofluorescence. Immunohistochemistry was done on 125 archived patient epithelial ovarian cancer tumors representing all epithelial ovarian cancer subtypes. T-cell factor-dependent transcription levels were determined in epithelial ovarian cancer cells using TOPflash/FOPflash in vivo assays. Phosphorothioated antisense oligonucleotides were transfected into cell lines and growth assayed by cell counting, anchorage-independent colony formation on soft agar, and xenografting into severe combined immunodeficient mice., Results: All six epithelial ovarian cancer cell lines and 82% of the patient samples overexpressed nuclear hPygo2 compared with control cells and benign disease. Depletion of hPygo2 by antisense oligonucleotides in both Wnt-active (TOV-112D) and Wnt-inactive serous (OVCAR-3, SKOV-3) and clear cell (TOV-21G) carcinoma cell lines halted growth, assessed using tissue culture, anchorage-independent, and xenograft assays., Conclusions: hPygo2 is unexpectedly widely expressed in, and required in the absence of, Wnt signaling for malignant growth of epithelial ovarian cancer, the deadliest gynecologic malignancy. These findings strongly suggest that inhibition of hPygo2 may be of therapeutic benefit for treating this disease.

We have identified two Xenopus mRNAs that encode proteins homologous to a component of the Wnt/beta-catenin transcriptional machinery known as Pygopus. The predicted proteins encoded by both mRNAs share the same structural properties with human Pygo-2, but with Xpygo-2alpha having an additional 21 N-terminal residues. Xpygo-2alpha messages accumulate in the prospective anterior neural plate after gastrulation and then are localized to the nervous system, rostral to and including the hindbrain. Xpygo-2beta mRNA is expressed in oocytes and early embryos but declines in level before and during gastrulation. In late neurula, Xpygo-2beta mRNA is restricted to the retinal field, including eye primordia and prospective forebrain. A C-terminal truncated mutant of Xpygo-2 containing the N-terminal Homology Domain (NHD) caused both axis duplication when injected at the 2-cell stage and inhibition of anterior neural development when injected in the prospective head, mimicking the previously described effects of Wnt-signaling activators. Inhibition of Xpygo-2alpha and Xpygo-2beta by injection of gene-specific antisense morpholino oligonucleotides into prospective anterior neurectoderm caused brain defects that were prevented by coinjection of Xpygo-2 mRNA. Both Xpygo-2alpha and Xpygo-2beta morpholinos reduced the eye and forebrain markers Xrx-1, Xpax-6, and XBF-1, while the Xpygo-2alpha morpholino also eliminated expression of the mid-hindbrain marker En-2. The differential expression and regulatory activities of Xpygo-2alpha/beta in rostral neural tissue indicate that they represent essential components of a novel mechanism for Wnt signaling in regionalization of the brain.

The head represents the most dorsal and anterior extent of the body axis. In Xenopus, the progressive determination of the head is an extremely complex process involving the activation and localized antagonism of a number of interdependent intracellular signaling pathways including the Wingless/Int-1 (Wnt), bone morphogenetic protein (BMP), and nodal-related pathways. The sequence of events that specify the head are: dorsal-ventral polarization and head organizer specification in the blastula; gastrulation; neural induction; and patterning of the anterior-posterior and dorsal-ventral neuraxes. Wnt signaling is required for the specification of the dorsal side initially but is then inhibited within the organizer once it has formed. Similarly, Wnt signaling is required along the length of the neural tube, but must be suppressed at its rostral end for normal brain development. Nodal signaling is also necessary for induction of the mesendoderm, but is subsequently suppressed in its dorsal-anterior extreme to specify head organizer. BMP signaling is required for ventral mesoderm and non-neural ectoderm, and must also be suppressed in the head organizer region and for the differentiation of the ventral midline of the neural tube. Thus, development of the head, and indeed the body plan in general, requires precisely timed and spatially restricted activation and repression of these signaling pathways.

The Rel/NF-kappa B gene family encodes a large group of transcriptional activators involved in myriad differentiation events, including embryonic development. We have shown previously that Xrel3, a Xenopus Rel/NF-kappa B-related gene, is expressed in the forebrain, dorsal aspect of the mid- and hindbrain, the otocysts and notochord of neurula and larval stage embryos. Overexpression of Xrel3 causes formation of embryonic tumours. We now show that Xrel3-induced tumours and animal caps from embryos injected with Xrel3 RNA express Otx2, Shh and Gli1. Heterodimerisation of a C-terminally deleted mutant of Xrel3 with wild-type Xrel3 inhibits in vitro binding of wild-type Xrel3 to Rel/NF-kappa B consensus DNA sequences. This dominant interference mutant disrupts Shh, Gli1 and Otx2 mRNA patterning and inhibits anterior development when expressed in the dorsal side of zygotes, which is rescued by co-injecting wild-type Xrel3 mRNA. In chick development, Rel activates Shh signalling, which is required for normal limb formation; Shh, Gli1 and Otx2 encode important neural patterning elements in vertebrates. The activation of these genes in tumours by Xrel3 overexpression and the inhibition of their expression and head development by a dominant interference mutant of Xrel3 indicates that Rel/NF-kappa B is required for activation of these genes and for anterior neural patterning in Xenopus.