Detection of gene cis-regulatory element perturbations in single-cell transcriptomes.

We introduce poly-adenine CRISPR gRNA-based single-cell RNA-sequencing (pAC-Seq), a method that enables the direct observation of guide RNAs (gRNAs) in scRNA-seq. We use pAC-Seq to assess the phenotypic consequences of CRISPR/Cas9 based alterations of gene cis-regulatory regions. We show that pAC-Seq is able to detect cis-regulatory-induced alteration of target gene expression even when biallelic loss of target gene expression occurs in only ~5% of cells. This low rate of biallelic loss significantly increases the number of cells required to detect the consequences of changes to the regulatory genome, but can be ameliorated by transcript-targeted sequencing. Based on our experimental results we model the power to detect regulatory genome induced transcriptomic effects based on the rate of mono/biallelic loss, baseline gene expression, and the number of cells per target gRNA.

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.

Nephronophthisis-associated CEP164 regulates cell cycle progression, apoptosis and epithelial-to-mesenchymal transition.

We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.

Mutations in LRRC50 predispose zebrafish and humans to seminomas.

Seminoma is a subclass of human testicular germ cell tumors (TGCT), the most frequently observed cancer in young men with a rising incidence. Here we describe the identification of a novel gene predisposing specifically to seminoma formation in a vertebrate model organism. Zebrafish carrying a heterozygous nonsense mutation in Leucine-Rich Repeat Containing protein 50 (lrrc50 also called dnaaf1), associated previously with ciliary function, are found to be highly susceptible to the formation of seminomas. Genotyping of these zebrafish tumors shows loss of heterozygosity (LOH) of the wild-type lrrc50 allele in 44.4% of tumor samples, correlating with tumor progression. In humans we identified heterozygous germline LRRC50 mutations in two different pedigrees with a family history of seminomas, resulting in a nonsense Arg488* change and a missense Thr590Met change, which show reduced expression of the wild-type allele in seminomas. Zebrafish in vivo complementation studies indicate the Thr590Met to be a loss-of-function mutation. Moreover, we show that a pathogenic Gln307Glu change is significantly enriched in individuals with seminoma tumors (13% of our cohort). Together, our study introduces an animal model for seminoma and suggests LRRC50 to be a novel tumor suppressor implicated in human seminoma pathogenesis.

Entpd5 is essential for skeletal mineralization and regulates phosphate homeostasis in zebrafish.

Bone mineralization is an essential step during the embryonic development of vertebrates, and bone serves vital functions in human physiology. To systematically identify unique gene functions essential for osteogenesis, we performed a forward genetic screen in zebrafish and isolated a mutant, no bone (nob), that does not form any mineralized bone. Positional cloning of nob identified the causative gene to encode ectonucleoside triphosphate/diphosphohydrolase 5 (entpd5); analysis of its expression pattern demonstrates that entpd5 is specifically expressed in osteoblasts. An additional mutant, dragonfish (dgf), exhibits ectopic mineralization in the craniofacial and axial skeleton and encodes a loss-of-function allele of ectonucleotide pyrophosphatase phosphodiesterase 1 (enpp1). Intriguingly, generation of double-mutant nob/dgf embryos restored skeletal mineralization in nob mutants, indicating that mechanistically, Entpd5 and Enpp1 act as reciprocal regulators of phosphate/pyrophosphate homeostasis in vivo. Consistent with this, entpd5 mutant embryos can be rescued by high levels of inorganic phosphate, and phosphate-regulating factors, such as fgf23 and npt2a, are significantly affected in entpd5 mutant embryos. Our study demonstrates that Entpd5 represents a previously unappreciated essential player in phosphate homeostasis and skeletal mineralization.

Macrophage-stimulating protein and calcium homeostasis in zebrafish.

To systematically identify novel gene functions essential for osteogenesis and skeletal mineralization, we performed a forward genetic mutagenesis screen in zebrafish and isolated a mutant that showed delayed skeletal mineralization. Analysis of the mutant phenotype in an osterix:nuclear-GFP transgenic background demonstrated that mutants contain osterix-expressing osteoblasts comparable to wild-type embryos. Positional cloning revealed a premature stop mutation in the macrophage-stimulating protein (msp) gene, predicted to result in a biologically inactive protein. Analysis of the embryonic expression pattern for the receptor for Msp, Ron, shows specific expression in the corpuscles of Stannius, a teleost-specific organ that produces stanniocalcin, a pivotal hormone in fish calcium homeostasis. Knockdown of Ron resulted in identical phenotypes as observed in msp mutants. Msp mutant embryos could be rescued by excess calcium. Consistent with a role for Msp/Ron in calcium homeostasis, calcium-regulating factors, such as pth1, pth2, stc1l, and trpv5/6 were significantly affected in msp mutant larvae. While Msp and Ron have previously been shown to play a critical role in a wide variety of biological processes, we introduce here the Msp/Ron signaling axis as a previously unappreciated player in calcium homeostasis and embryonic skeletal mineralization.

Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia.

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lineages. Hematopoietic tissues are highly proliferative, with enlarged populations of c-myb(+) hematopoietic stem cells and circulating erythroid precursors. Chemical activation of hypoxia-inducible factor signaling recapitulated aspects of the vhl(-/-) phenotype. Furthermore, microarray expression analysis confirms the hypoxic response and hematopoietic phenotype observed in vhl(-/-) embryos. We conclude that VHL participates in regulating hematopoiesis and erythroid differentiation. Injections with human VHLp30 and R200W mutant mRNA demonstrate functional conservation of VHL between mammals and zebrafish at the amino acid level, indicating that vhl mutants are a powerful new tool to study genotype-phenotype correlations in human disease. Zebrafish vhl mutants are the first congenital embryonic viable systemic vertebrate animal model for VHL, representing the most accurate model for VHL-associated polycythemia to date. They will contribute to our understanding of hypoxic signaling, hematopoiesis, and VHL-associated disease progression.

mRNA structural dynamics shape Argonaute-target interactions.

Small interfering RNAs (siRNAs) promote RNA degradation in a variety of processes and have important clinical applications. siRNAs direct cleavage of target RNAs by guiding Argonaute2 (AGO2) to its target site. Target site accessibility is critical for AGO2-target interactions, but how target site accessibility is controlled in vivo is poorly understood. Here, we use live-cell single-molecule imaging in human cells to determine rate constants of the AGO2 cleavage cycle in vivo. We find that the rate-limiting step in mRNA cleavage frequently involves unmasking of target sites by translating ribosomes. Target site masking is caused by heterogeneous intramolecular RNA-RNA interactions, which can conceal target sites for many minutes in the absence of translation. Our results uncover how dynamic changes in mRNA structure shape AGO2-target recognition, provide estimates of mRNA folding and unfolding rates in vivo, and provide experimental evidence for the role of mRNA structural dynamics in control of mRNA-protein interactions.

Control of Angiogenesis via a VHL/miR-212/132 Axis.

A common feature of tumorigenesis is the upregulation of angiogenesis pathways in order to supply nutrients via the blood for the growing tumor. Understanding how cells promote angiogenesis and how to control these processes pharmaceutically are of great clinical interest. Clear cell renal cell carcinoma (ccRCC) is the most common form of sporadic and inherited kidney cancer which is associated with excess neovascularization. ccRCC is highly associated with biallelic mutations in the von Hippel-Lindau (VHL) tumor suppressor gene. Although upregulation of the miR-212/132 family and disturbed VHL signaling have both been linked with angiogenesis, no evidence of a possible connection between the two has yet been made. We show that miRNA-212/132 levels are increased after loss of functional pVHL, the protein product of the VHL gene, in vivo and in vitro. Furthermore, we show that blocking miRNA-212/132 with anti-miRs can significantly alleviate the excessive vascular branching phenotype characteristic of vhl-/- mutant zebrafish. Moreover, using human umbilical vascular endothelial cells (HUVECs) and an endothelial cell/pericyte coculture system, we observed that VHL knockdown promotes endothelial cells neovascularization capacity in vitro, an effect which can be inhibited by anti-miR-212/132 treatment. Taken together, our results demonstrate an important role for miRNA-212/132 in angiogenesis induced by loss of VHL. Intriguingly, this also presents a possibility for the pharmaceutical manipulation of angiogenesis by modulating levels of MiR212/132.

The von Hippel-Lindau Gene Is Required to Maintain Renal Proximal Tubule and Glomerulus Integrity in Zebrafish Larvae.

BACKGROUND: von Hippel-Lindau (VHL) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine Vhl models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human VHL gene. METHODS: We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in vhl mutant zebrafish, supported by human cell culture assays. RESULTS: Here, we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. vhl mutant glomeruli are enlarged, cxcr4a+ capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the vhl-/- tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1-43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the vhl mutant pronephric tubule, which we can recapitulate in human cells lacking VHL. CONCLUSIONS: Our data indicates that vhl is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.

TCF21 hypermethylation regulates renal tumor cell clonogenic proliferation and migration.

We recently identified hypermethylation at the gene promoter of transcription factor 21 (TCF21) in clear cell sarcoma of the kidney (CCSK), a rare pediatric renal tumor. TCF21 is a transcription factor involved in tubular epithelial development of the kidney and is a candidate tumor suppressor. As there are no in vitro models of CCSK, we employed a well-established clear cell renal cell carcinoma (ccRCC) cell line, 786-O, which also manifests high methylation at the TCF21 promoter, with consequent low TCF21 expression. The tumor suppressor function of TCF21 has not been functionally addressed in ccRCC cells; we aimed to explore the functional potential of TCF21 expression in ccRCC cells in vitro. 786-O clones stably transfected with either pBABE-TCF21-HA construct or pBABE vector alone were functionally analyzed. We found that ectopic expression of TCF21 in 786-O cells results in a trend toward decreased cell proliferation (not significant) and significantly decreased migration compared with mock-transfected 786-O cells. Although the number of colonies established in colony formation assays was not different between 786-O clones, colony size was significantly reduced in 786-O cells expressing TCF21. To investigate whether the changes in migration were due to epithelial-to-mesenchymal transition changes, we interrogated the expression of selected epithelial and mesenchymal markers. Although we observed upregulation of mRNA and protein levels of epithelial marker E-cadherin in clones overexpressing TCF21, this did not result in surface expression of E-cadherin as measured by fluorescence-activated cell sorting and immunofluorescence. Furthermore, mRNA expression of the mesenchymal markers vimentin (VIM) and SNAI1 was not significantly decreased in TCF21-expressing 786-O cells, while protein levels of VIM were markedly decreased. We conclude that re-expression of TCF21 in renal cancer cells that have silenced their endogenous TCF21 locus through hypermethylation results in reduced clonogenic proliferation, reduced migration, and reduced mesenchymal-like characteristics, suggesting a tumor suppressor function for transcription factor 21.

Segmentation of the zebrafish axial skeleton relies on notochord sheath cells and not on the segmentation clock.

Segmentation of the axial skeleton in amniotes depends on the segmentation clock, which patterns the paraxial mesoderm and the sclerotome. While the segmentation clock clearly operates in teleosts, the role of the sclerotome in establishing the axial skeleton is unclear. We severely disrupt zebrafish paraxial segmentation, yet observe a largely normal segmentation process of the chordacentra. We demonstrate that axial entpd5+ notochord sheath cells are responsible for chordacentrum mineralization, and serve as a marker for axial segmentation. While autonomous within the notochord sheath, entpd5 expression and centrum formation show some plasticity and can respond to myotome pattern. These observations reveal for the first time the dynamics of notochord segmentation in a teleost, and are consistent with an autonomous patterning mechanism that is influenced, but not determined by adjacent paraxial mesoderm. This behavior is not consistent with a clock-type mechanism in the notochord.

Non-invasive sources of cells with primary cilia from pediatric and adult patients.

BACKGROUND: Ciliopathies give rise to a multitude of organ-specific pathologies; obtaining relevant primary patient material is useful for both diagnostics and research. However, acquisition of primary ciliated cells from patients, particularly pediatric patients, presents multiple difficulties. Biopsies and blood samples are invasive, and patients (and their parents) may be reluctant to travel to medical centers, especially for research purposes. We sought to develop non-invasive methods of obtaining viable and ciliated primary cells from ciliopathy patients which could be obtained in the home environment. FINDINGS: We introduce two methods for the non-invasive acquisition of primary ciliated cells. In one approach, we collected spontaneously shed deciduous (milk) teeth from children. Fibroblast-like cells were observed after approximately 2 weeks of culture of fragmented teeth. Secondly, urine samples were collected from children or adults. Cellular content was isolated and after approximately 1 week, renal epithelial cells were observed. Both urine and tooth-derived cells ciliate and express ciliary proteins visible with immunofluorescence. Urine-derived renal epithelial cells (URECs) are amenable to 3D culturing, siRNA knockdown, and ex vivo drug testing. CONCLUSIONS: As evidence continues to accumulate showing that the primary cilium has a central role in development and disease, the need for readily available and ciliated patient cells will increase. Here, we introduce two methods for the non-invasive acquisition of cells with primary cilia. We believe that these cells can be used for further ex vivo study of ciliopathies and in the future, for personalized medicine.

A zebrafish model for VHL and hypoxia signaling.

The von Hippel-Lindau (VHL) tumor suppressor gene encodes an adaptor protein that regulates an array of transcription-dependent and -independent cellular and physiological processes. Mutations in this gene cause VHL disease, congenital polycythemia, and several sporadic tumor types. The last 15 years of fundamental and clinical research have helped define the phenotypic spectrum of VHL-associated diseases and have introduced new cellular functions for pVHL. Here, we review the current knowledge of VHL function, and the different animal models for VHL disease, with a particular focus on the zebrafish. Zebrafish vhl mutants develop key aspects of the human disease condition, including activation of the hypoxia-inducible factor (HIF) signaling pathway, polycythemia, excessive neovascularization, macular edema, and pronephric abnormalities. The zebrafish vhl model offers a platform for the identification of genetic pathways, modifiers, and interactors involved in the development of VHL-associated neoplasms. Vhl mutants represent a unique and clinically relevant in vivo model for studying genotype-phenotype correlations and the identification of prognostic biomarkers. The amenability of zebrafish for chemical genetic screens will not only be helpful to identify novel therapeutic agents but may also reveal novel processes that require regulation by VHL.

von Hippel-Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish.

Biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene predisposes human patients to the development of highly vascularized neoplasms in multiple organ systems. We show that zebrafish vhl mutants display a marked increase in blood vessel formation throughout the embryo, starting at 2 days post-fertilization. The most severe neovascularization is observed in distinct areas that overlap with high vegfa mRNA expression, including the vhl mutant brain and eye. Real-time quantitative PCR revealed increased expression of the duplicated VEGFA orthologs vegfaa and vegfab, and of vegfb and its receptors flt1, kdr and kdr-like, indicating increased vascular endothelial growth factor (Vegf) signaling in vhl mutants. Similar to VHL-associated retinal neoplasms, diabetic retinopathy and age-related macular degeneration, we show, by tetramethyl rhodamine-dextran angiography, that vascular abnormalities in the vhl(-/-) retina lead to vascular leakage, severe macular edema and retinal detachment. Significantly, vessels in the brain and eye express cxcr4a, a marker gene expressed by tumor and vascular cells in VHL-associated hemangioblastomas and renal cell carcinomas. VEGF receptor (VEGFR) tyrosine kinase inhibition (through exposure to sunitinib and 676475) blocked vhl(-/-)-induced angiogenesis in all affected tissues, demonstrating that Vegfaa, Vegfab and Vegfb are key effectors of the vhl(-/-) angiogenic phenotype through Flt1, Kdr and Kdr-like signaling. Since we show that the vhl(-/-) angiogenic phenotype shares distinct characteristics with VHL-associated vascular neoplasms, zebrafish vhl mutants provide a valuable in vivo vertebrate model to elucidate underlying mechanisms contributing to the development of these lesions. Furthermore, vhl mutant zebrafish embryos carrying blood vessel-specific transgenes represent a unique and clinically relevant model for tissue-specific, hypoxia-induced pathological angiogenesis and vascular retinopathies. Importantly, they will allow for a cost-effective, non-invasive and efficient way to screen for novel pharmacological agents and combinatorial treatments.