Human prostate organoid generation and the identification of prostate development drivers using inductive rodent tissues.

The reactivation of developmental genes and pathways during adulthood may contribute to pathogenesis of diseases such as prostate cancer. Analysis of the mechanistic links between development and disease could be exploited to identify signalling pathways leading to disease in the prostate. However, the mechanisms underpinning prostate development require further characterisation to interrogate fully the link between development and disease. Previously, our group developed methods to produce prostate organoids using induced pluripotent stem cells (iPSCs). Here, we show that human iPSCs can be differentiated into prostate organoids using neonatal rat seminal vesicle mesenchyme in vitro. The organoids can be used to study prostate development or modified to study prostate cancer. We also elucidated molecular drivers of prostate induction through RNA-sequencing analyses of the rat urogenital sinus and neonatal seminal vesicles. We identified candidate drivers of prostate development evident in the inductive mesenchyme and epithelium involved with prostate specification. Our top candidates included Spx, Trib3, Snai1, Snai2, Nrg2 and Lrp4. This work lays the foundations for further interrogation of the reactivation of developmental genes in adulthood, leading to prostate disease.

Mouse genetics reveals Barttin as a genetic modifier of Joubert syndrome.

Genetic and phenotypic heterogeneity and the lack of sufficiently large patient cohorts pose a significant challenge to understanding genetic associations in rare disease. Here we identify Bsnd (alias Barttin) as a genetic modifier of cystic kidney disease in Joubert syndrome, using a Cep290-deficient mouse model to recapitulate the phenotypic variability observed in patients by mixing genetic backgrounds in a controlled manner and performing genome-wide analysis of these mice. Experimental down-regulation of Bsnd in the parental mouse strain phenocopied the severe cystic kidney phenotype. A common polymorphism within human BSND significantly associates with kidney disease severity in a patient cohort with CEP290 mutations. The striking phenotypic modifications we describe are a timely reminder of the value of mouse models and highlight the significant contribution of genetic background. Furthermore, if appropriately managed, this can be exploited as a powerful tool to elucidate mechanisms underlying human disease heterogeneity.

Targeted exon skipping rescues ciliary protein composition defects in Joubert syndrome patient fibroblasts.

Joubert syndrome (JBTS) is an incurable multisystem ciliopathy syndrome. The most commonly mutated gene in JBTS patients with a cerebello-retinal-renal phenotype is CEP290 (alias JBTS5). The encoded CEP290 protein localises to the proximal end of the primary cilium, in the transition zone, where it controls ciliary protein composition and signalling. We examined primary cilium structure and composition in fibroblast cells derived from homozygous and compound heterozygous JBTS5 patients with nonsense mutations in CEP290 and show that elongation of cilia, impaired ciliogenesis and ciliary composition defects are typical features in JBTS5 cells. Targeted skipping of the mutated exon c.5668 G > T using antisense oligonucleotide (ASO) therapy leads to restoration of CEP290 protein expression and functions at the transition zone in homozygous and compound heterozygous JBTS5 cells, allowing a rescue of both cilia morphology and ciliary composition. This study, by demonstrating that targeted exon skipping is able to rescue ciliary protein composition defects, provides functional evidence for the efficacy of this approach in the treatment of JBTS.

Targeted exon skipping of a CEP290 mutation rescues Joubert syndrome phenotypes in vitro and in a murine model.

Genetic treatments of renal ciliopathies leading to cystic kidney disease would provide a real advance in current therapies. Mutations in CEP290 underlie a ciliopathy called Joubert syndrome (JBTS). Human disease phenotypes include cerebral, retinal, and renal disease, which typically progresses to end stage renal failure (ESRF) within the first two decades of life. While currently incurable, there is often a period of years between diagnosis and ESRF that provides a potential window for therapeutic intervention. By studying patient biopsies, patient-derived kidney cells, and a mouse model, we identify abnormal elongation of primary cilia as a key pathophysiological feature of CEP290-associated JBTS and show that antisense oligonucleotide (ASO)-induced splicing of the mutated exon (41, G1890*) restores protein expression in patient cells. We demonstrate that ASO-induced splicing leading to exon skipping is tolerated, resulting in correct localization of CEP290 protein to the ciliary transition zone, and restoration of normal cilia length in patient kidney cells. Using a gene trap Cep290 mouse model of JBTS, we show that systemic ASO treatment can reduce the cystic burden of diseased kidneys in vivo. These findings indicate that ASO treatment may represent a promising therapeutic approach for kidney disease in CEP290-associated ciliopathy syndromes.

ARL3 Mutations Cause Joubert Syndrome by Disrupting Ciliary Protein Composition.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

Human urine-derived renal epithelial cells provide insights into kidney-specific alternate splicing variants.

The majority of multi-exon genes undergo alternative splicing to produce different mRNA transcripts and this may occur in a tissue-specific manner. Assessment of mRNA transcripts isolated from blood samples may sometimes be unhelpful in determining the affect on function of putative splice-site variants affecting kidney-specific mRNA transcripts. Here we present data demonstrating the power of using human urine-derived renal epithelial cells (hUREC) as a source of kidney RNA. We report clinical and molecular genetic data from three affected cases from two families all with end-stage renal disease by 15 years of age. In both families, heterozygous variants which are predicted to effect function in NPHP3 were found on one allele, in combination with a synonymous SNV (c.2154C>T; p.Phe718=), 18 base pairs from the exon-intron boundary within exon 15 of NPHP3. The only mRNA transcript amplified from wild-type whole blood showed complete splicing out of exon 15. Urine samples obtained from control subjects and the father of family 2, who carried the synonymous SNV variant, were therefore used to culture hUREC and allowed us to obtain kidney-specific mRNA. Control kidney mRNA showed retention of exon 15, while the mRNA from the patient's father confirmed evidence of a heterozygous alternate splicing of exon 15 of NPHP3. Analysis of RNA derived from hUREC allows for a comparison of kidney-specific and whole-blood RNA transcripts and for assessment of the effect on function of putative splice variants leading to end-stage kidney disease.

A human patient-derived cellular model of Joubert syndrome reveals ciliary defects which can be rescued with targeted therapies.

Joubert syndrome (JBTS) is the archetypal ciliopathy caused by mutation of genes encoding ciliary proteins leading to multi-system phenotypes, including a cerebello-retinal-renal syndrome. JBTS is genetically heterogeneous, however mutations in CEP290 are a common underlying cause. The renal manifestation of JBTS is a juvenile-onset cystic kidney disease, known as nephronophthisis, typically progressing to end-stage renal failure within the first two decades of life, thus providing a potential window for therapeutic intervention. In order to increase understanding of JBTS and its associated kidney disease and to explore potential treatments, we conducted a comprehensive analysis of primary renal epithelial cells directly isolated from patient urine (human urine-derived renal epithelial cells, hURECs). We demonstrate that hURECs from a JBTS patient with renal disease have elongated and disorganized primary cilia and that this ciliary phenotype is specifically associated with an absence of CEP290 protein. Treatment with the Sonic hedgehog (Shh) pathway agonist purmorphamine or cyclin-dependent kinase inhibition (using roscovitine and siRNA directed towards cyclin-dependent kinase 5) ameliorated the cilia phenotype. In addition, purmorphamine treatment was shown to reduce cyclin-dependent kinase 5 in patient cells, suggesting a convergence of these signalling pathways. To our knowledge, this is the most extensive analysis of primary renal epithelial cells from JBTS patients to date. It demonstrates the feasibility and power of this approach to directly assess the consequences of patient-specific mutations in a physiologically relevant context and a previously unrecognized convergence of Shh agonism and cyclin-dependent kinase inhibition as potential therapeutic targets.

Murine Joubert syndrome reveals Hedgehog signaling defects as a potential therapeutic target for nephronophthisis.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

WT1 regulates murine hematopoiesis via maintenance of VEGF isoform ratio.

Mutations in the Wilms tumor suppressor 1 (WT1) gene are as frequent in acute myeloid leukemia (AML) as in nephroblastma and predict poor prognosis. However, the role of WT1 in hematopoiesis remains unclear. We show that Wt1-deficient mouse embryonic stem cells exhibit reduced hematopoietic potential caused by vascular endothelial growth factor A (Vegf-a)-dependent apoptosis of hematopoietic progenitor cells associated with overproduction of the Vegf-a120 isoform. We demonstrate that Wt1 promotes exon inclusion using a Vegf-a minigene-based splicing assay. These data identify a critical role for Wt1 in hematopoiesis and Vegf-a as a cellular RNA whose splicing is potentially regulated by Wt1. The correction of Wt1 deficiency by treatment with exogenous Vegf-a protein indicates that the Wt1/Vegf-a axis is a molecular pathway that could be exploited for the management/treatment of poor prognosis AMLs.

Effects on kidney disease, fertility and development in mice inheriting a protein-truncating Denys-Drash syndrome allele (Wt1tmT396).

Denys-Drash syndrome (DDS) is caused by heterozygous mutations of the Wilms' tumour suppressor gene, WT1, characterised by early-onset diffuse mesangial sclerosis often associated with male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that the Wt1tmT396 allele induces DDS kidney disease in mice. In the present study heterozygotes (Wt1tmT396/+) were generated on inbred (129/Ola), crossbred (B6/129) and MF1 second backcross (MF1-N2) backgrounds. Whereas male heterozygotes on each background were fertile, inbred heterozygous females were infertile. Kidney disease (proteinuria and sclerosis) was not congenital and developed significantly earlier in inbred mice, although with variable onset. Disease onset in MF1-N2 stocks occurred later in Wt1tmT396/+ mice than reported previously for Wt1R394W/+ mice, and while no kidney disease has been reported in B6/129 Wt1+/- mice, B6/129 Wt1tmT396/+ mice were affected. Offspring of both male and female B6/129 and MF1-N2 Wt1tmT396/+ mice developed kidney disease, but its incidence was significantly higher in offspring of female heterozygotes. Wt1tmT396/tmT396 embryos exhibited identical developmental abnormalities to those reported for Wt1-/- embryos. The results indicate that the Wt1 (tmT396) allele does not predispose to Wilms' tumourigenesis or male pseudohermaphroditism, its effect on kidney disease and female fertility depends on genetic background, stochastic factors may affect disease onset, and disease transmission is subject to a partial parent-of-origin effect. Since the Wt1tmT396 allele has no detectable intrinsic functional activity in vivo, and kidney disease progression is affected by the type of Wt1 mutation, the data support the view that DDS nephropathy results from a dominant-negative action rather than WT1 haploinsufficiency or gain-of-function.

K-ras proto-oncogene exhibits tumor suppressor activity as its absence promotes tumorigenesis in murine teratomas.

Ras proteins transduce signals from membrane-bound receptors via multiple downstream effector pathways and thereby affect fundamental cellular processes, including proliferation, apoptosis, and differentiation. K-ras activating mutations play a key role in neoplastic progression and are particularly prevalent in colorectal, pancreatic, and lung cancers. The present study addressed whether the K-ras proto-oncogene displays a tumor suppressor function by comparative analysis of mouse teratomas derived from wild-type embryonic stem (ES) cells, K-ras null (K-ras(-/-)) ES cells, and K-ras(-/-) ES cells that stably reexpress either wild-type K-ras(gly12) or oncogenic K-ras(val12). K-ras(-/-) and K-ras(val12) teratomas were significantly larger than teratomas that expressed wild-type K-ras, contained significantly higher proportions of undifferentiated embryonal carcinoma-like cells, and showed significantly increased mitotic activity. However, K-ras(val12) but not K-ras(-/-) teratomas exhibited significantly higher levels of apoptosis than wild-type teratomas. K-ras(-/-) and K-ras(val12) ES cells showed a higher capacity for stem cell self-renewal in vitro compared with wild-type ES cells, and reexpression of K-ras(gly12) in K-ras(-/-) ES cells restored the K-ras(-/-) phenotype to wild-type values. Thus, in view of evidence that tumors can derive from tissue stem cells and that tumors harbor "cancer stem cells," aberrant K-ras expression could promote neoplastic progression by increasing their capacity for self-renewal.

Murine Denys-Drash syndrome: evidence of podocyte de-differentiation and systemic mediation of glomerulosclerosis.

Denys-Drash syndrome (DDS) is caused by dominant mutations of the Wilms' tumour suppressor gene, WT1, and characterized by a nephropathy involving diffuse mesangial sclerosis, male pseudohermaphroditism and/or Wilms' tumourigenesis. Previously, we reported that heterozygosity for the Wt1tmT396 mutation induces DDS in heterozygous and chimeric (Wt1tmT396/+<-->+/+) mice. In the present study, the fate of Wt1 mutant cells in chimeric kidneys was assessed by in situ marker analysis, and immunocytochemistry was used to re-examine the claim that glomerulosclerosis (GS) is caused by loss of WT1 and persistent Pax-2 expression by podocytes. Wt1 mutant cells colonized glomeruli efficiently, including podocytes, but some sclerotic glomeruli contained no detectable Wt1 mutant cells. The development of GS was preceded by widespread loss of ZO-1 signal in podocytes (even in kidneys where <5% of glomeruli contained Wt1 mutant podocytes), increased intra-renal renin expression, and de novo podocyte TGF-beta1 expression, but not podocyte Pax-2 expression or loss of WT1, synaptopodin, alpha-actinin-4 or nephrin expression. However, podocytes in partially sclerotic glomeruli that still expressed WT1 at high levels showed reduced vimentin expression, cell cycle re-entry, and re-expressed desmin, cytokeratin and Pax-2. The results suggest that: (i) GS is not due to loss of WT1 expression by podocytes; (ii) podocyte Pax-2 expression reflects re-expression rather than persistent expression, and is the consequence of GS; (iii) GS is mediated systemically and the mechanism involves activation of the renin-angiotensin system; and (iv) podocytes undergo typical maturational changes but subsequently de-differentiate and revert to an immature phenotype during disease progression.

Mice lacking the 68-amino-acid, mammal-specific N-terminal extension of WT1 develop normally and are fertile.

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.