Implication of transcription factor FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below age 30 years. Many monogenic forms have been discovered due to comprehensive genetic testing like exome sequencing. However, disease-causing variants in known disease-associated genes only explain a proportion of cases. Here, we aim to unravel underlying molecular mechanisms of syndromic CAKUT in three unrelated multiplex families with presumed autosomal recessive inheritance. Exome sequencing in the index individuals revealed three different rare homozygous variants in FOXD2, encoding a transcription factor not previously implicated in CAKUT in humans: a frameshift in the Arabic and a missense variant each in the Turkish and the Israeli family with segregation patterns consistent with autosomal recessive inheritance. CRISPR/Cas9-derived Foxd2 knockout mice presented with a bilateral dilated kidney pelvis accompanied by atrophy of the kidney papilla and mandibular, ophthalmologic, and behavioral anomalies, recapitulating the human phenotype. In a complementary approach to study pathomechanisms of FOXD2-dysfunction-mediated developmental kidney defects, we generated CRISPR/Cas9-mediated knockout of Foxd2 in ureteric bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important for kidney/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a shift toward a stromal cell identity. Histology of Foxd2 knockout mouse kidneys confirmed increased fibrosis. Further, genome-wide association studies suggest that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Thus, our studies help in genetic diagnostics of monogenic CAKUT and in understanding of monogenic and multifactorial kidney diseases.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

Knockout mouse models as a resource for the study of rare diseases.

Rare diseases (RDs) are a challenge for medicine due to their heterogeneous clinical manifestations and low prevalence. There is a lack of specific treatments and only a few hundred of the approximately 7,000 RDs have an approved regime. Rapid technological development in genome sequencing enables the mass identification of potential candidates that in their mutated form could trigger diseases but are often not confirmed to be causal. Knockout (KO) mouse models are essential to understand the causality of genes by allowing highly standardized research into the pathogenesis of diseases. The German Mouse Clinic (GMC) is one of the pioneers in mouse research and successfully uses (preclinical) data obtained from single-gene KO mutants for research into monogenic RDs. As part of the International Mouse Phenotyping Consortium (IMPC) and INFRAFRONTIER, the pan-European consortium for modeling human diseases, the GMC expands these preclinical data toward global collaborative approaches with researchers, clinicians, and patient groups.Here, we highlight proprietary genes that when deleted mimic clinical phenotypes associated with known RD targets (Nacc1, Bach2, Klotho alpha). We focus on recognized RD genes with no pre-existing KO mouse models (Kansl1l, Acsf3, Pcdhgb2, Rabgap1, Cox7a2) which highlight novel phenotypes capable of optimizing clinical diagnosis. In addition, we present genes with intriguing phenotypic data (Zdhhc5, Wsb2) that are not presently associated with known human RDs.This report provides comprehensive evidence for genes that when deleted cause differences in the KO mouse across multiple organs, providing a huge translational potential for further understanding monogenic RDs and their clinical spectrum. Genetic KO studies in mice are valuable to further explore the underlying physiological mechanisms and their overall therapeutic potential.

AOX delays the onset of the lethal phenotype in a mouse model of Uqcrh (complex III) disease.

The alternative oxidase, AOX, provides a by-pass of the cytochrome segment of the mitochondrial respiratory chain when the chain is unavailable. AOX is absent from mammals, but AOX from Ciona intestinalis is benign when expressed in mice. Although non-protonmotive, so does not contribute directly to ATP production, it has been shown to modify and in some cases rescue phenotypes of respiratory-chain disease models. Here we studied the effect of C. intestinalis AOX on mice engineered to express a disease-equivalent mutant of Uqcrh, encoding the hinge subunit of mitochondrial respiratory complex III, which results in a complex metabolic phenotype beginning at 4-5 weeks, rapidly progressing to lethality within a further 6-7 weeks. AOX expression delayed the onset of this phenotype by several weeks, but provided no long-term benefit. We discuss the significance of this finding in light of the known and hypothesized effects of AOX on metabolism, redox homeostasis, oxidative stress and cell signaling. Although not a panacea, the ability of AOX to mitigate disease onset and progression means it could be useful in treatment.

Implication of FOXD2 dysfunction in syndromic congenital anomalies of the kidney and urinary tract (CAKUT).

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause for chronic kidney disease below 30 years of age. Many monogenic forms have been discovered mainly due to comprehensive genetic testing like exome sequencing (ES). However, disease-causing variants in known disease-associated genes still only explain a proportion of cases. Aim of this study was to unravel the underlying molecular mechanism of syndromic CAKUT in two multiplex families with presumed autosomal recessive inheritance. Methods and Results: ES in the index individuals revealed two different rare homozygous variants in FOXD2, a transcription factor not previously implicated in CAKUT in humans: a frameshift in family 1 and a missense variant in family 2 with family segregation patterns consistent with autosomal-recessive inheritance. CRISPR/Cas9-derived Foxd2 knock-out (KO) mice presented with bilateral dilated renal pelvis accompanied by renal papilla atrophy while extrarenal features included mandibular, ophthalmologic, and behavioral anomalies, recapitulating the phenotype of humans with FOXD2 dysfunction. To study the pathomechanism of FOXD2-dysfunction-mediated developmental renal defects, in a complementary approach, we generated CRISPR/Cas9-mediated KO of Foxd2 in ureteric-bud-induced mouse metanephric mesenchyme cells. Transcriptomic analyses revealed enrichment of numerous differentially expressed genes important in renal/urogenital development, including Pax2 and Wnt4 as well as gene expression changes indicating a cell identity shift towards a stromal cell identity. Histology of Foxd2 KO mouse kidneys confirmed increased fibrosis. Further, GWAS data (genome-wide association studies) suggests that FOXD2 could play a role for maintenance of podocyte integrity during adulthood. Conclusions: In summary, our data implicate that FOXD2 dysfunction is a very rare cause of autosomal recessive syndromic CAKUT and suggest disturbances of the PAX2-WNT4 cell signaling axis contribute to this phenotype.

Knockout of the Complex III subunit Uqcrh causes bioenergetic impairment and cardiac contractile dysfunction.

Ubiquinol cytochrome c reductase hinge protein (UQCRH) is required for the electron transfer between cytochrome c1 and c of the mitochondrial cytochrome bc1 Complex (CIII). A two-exon deletion in the human UQCRH gene has recently been identified as the cause for a rare familial mitochondrial disorder. Deletion of the corresponding gene in the mouse (Uqcrh-KO) resulted in striking biochemical and clinical similarities including impairment of CIII, failure to thrive, elevated blood glucose levels, and early death. Here, we set out to test how global ablation of the murine Uqcrh affects cardiac morphology and contractility, and bioenergetics. Hearts from Uqcrh-KO mutant mice appeared macroscopically considerably smaller compared to wildtype littermate controls despite similar geometries as confirmed by transthoracic echocardiography (TTE). Relating TTE-assessed heart to body mass revealed the development of subtle cardiac enlargement, but histopathological analysis showed no excess collagen deposition. Nonetheless, Uqcrh-KO hearts developed pronounced contractile dysfunction. To assess mitochondrial functions, we used the high-resolution respirometer NextGen-O2k allowing measurement of mitochondrial respiratory capacity through the electron transfer system (ETS) simultaneously with the redox state of ETS-reactive coenzyme Q (Q), or production of reactive oxygen species (ROS). Compared to wildtype littermate controls, we found decreased mitochondrial respiratory capacity and more reduced Q in Uqcrh-KO, indicative for an impaired ETS. Yet, mitochondrial ROS production was not generally increased. Taken together, our data suggest that Uqcrh-KO leads to cardiac contractile dysfunction at 9 weeks of age, which is associated with impaired bioenergetics but not with mitochondrial ROS production. Global ablation of the Uqcrh gene results in functional impairment of CIII associated with metabolic dysfunction and postnatal developmental arrest immediately after weaning from the mother. Uqcrh-KO mice show dramatically elevated blood glucose levels and decreased ability of isolated cardiac mitochondria to consume oxygen (O2). Impaired development (failure to thrive) after weaning manifests as a deficiency in the gain of body mass and growth of internal organ including the heart. The relative heart mass seemingly increases when organ mass calculated from transthoracic echocardiography (TTE) is normalized to body mass. Notably, the heart shows no signs of collagen deposition, yet does develop a contractile dysfunction reflected by a decrease in ejection fraction and fractional shortening.

Post-synaptic scaffold protein TANC2 in psychiatric and somatic disease risk.

Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2)-disrupting variants were disease causing in NDD patients. The post-synaptic scaffold protein TANC2 is essential for dendrite formation in synaptic plasticity and plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2-disrupted function model in which mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain, such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility, underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.

Characterising a homozygous two-exon deletion in UQCRH: comparing human and mouse phenotypes.

Mitochondrial disorders are clinically and genetically diverse, with isolated complex III (CIII) deficiency being relatively rare. Here, we describe two affected cousins, presenting with recurrent episodes of severe lactic acidosis, hyperammonaemia, hypoglycaemia and encephalopathy. Genetic investigations in both cases identified a homozygous deletion of exons 2 and 3 of UQCRH, which encodes a structural complex III (CIII) subunit. We generated a mouse model with the equivalent homozygous Uqcrh deletion (Uqcrh-/- ), which also presented with lactic acidosis and hyperammonaemia, but had a more severe, non-episodic phenotype, resulting in failure to thrive and early death. The biochemical phenotypes observed in patient and Uqcrh-/- mouse tissues were remarkably similar, displaying impaired CIII activity, decreased molecular weight of fully assembled holoenzyme and an increase of an unexpected large supercomplex (SXL ), comprising mostly of one complex I (CI) dimer and one CIII dimer. This phenotypic similarity along with lentiviral rescue experiments in patient fibroblasts verifies the pathogenicity of the shared genetic defect, demonstrating that the Uqcrh-/- mouse is a valuable model for future studies of human CIII deficiency.

A comprehensive phenotypic characterization of a whole-body Wdr45 knock-out mouse.

Pathogenic variants in the WDR45 (OMIM: 300,526) gene on chromosome Xp11 are the genetic cause of a rare neurological disorder characterized by increased iron deposition in the basal ganglia. As WDR45 encodes a beta-propeller scaffold protein with a putative role in autophagy, the disease has been named Beta-Propeller Protein-Associated Neurodegeneration (BPAN). BPAN represents one of the four most common forms of Neurodegeneration with Brain Iron Accumulation (NBIA). In the current study, we generated and characterized a whole-body Wdr45 knock-out (KO) mouse model. The model, developed using TALENs, presents a 20-bp deletion in exon 2 of Wdr45. Homozygous females and hemizygous males are viable, proving that systemic depletion of Wdr45 does not impair viability and male fertility in mice. The in-depth phenotypic characterization of the mouse model revealed neuropathology signs at four months of age, neurodegeneration progressing with ageing, hearing and visual impairment, specific haematological alterations, but no brain iron accumulation. Biochemically, Wdr45 KO mice presented with decreased complex I (CI) activity in the brain, suggesting that mitochondrial dysfunction accompanies Wdr45 deficiency. Overall, the systemic Wdr45 KO described here complements the two mouse models previously reported in the literature (PMIDs: 26,000,824, 31,204,559) and represents an additional robust model to investigate the pathophysiology of BPAN and to test therapeutic strategies for the disease.

Imbalances in the eye lens proteome are linked to cataract formation.

The prevalent model for cataract formation in the eye lens posits that damaged crystallin proteins form light-scattering aggregates. The α-crystallins are thought to counteract this process as chaperones by sequestering misfolded crystallin proteins. In this scenario, chaperone pool depletion would result in lens opacification. Here we analyze lenses from different mouse strains that develop early-onset cataract due to point mutations in α-, ß-, or γ-crystallin proteins. We find that these mutant crystallins are unstable in vitro; in the lens, their levels are substantially reduced, and they do not accumulate in the water-insoluble fraction. Instead, all the other crystallin proteins, including the α-crystallins, are found to precipitate. The changes in protein composition and spatial organization of the crystallins observed in the mutant lenses suggest that the imbalance in the lenticular proteome and altered crystallin interactions are the bases for cataract formation, rather than the aggregation propensity of the mutant crystallins.

The rRNA m6A methyltransferase METTL5 is involved in pluripotency and developmental programs.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N6-methyladenosine (m6A) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes m6A in 18S rRNA at position A1832 We report that absence of Mettl5 in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in METTL5, thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of m6A in rRNA in stemness, differentiation, development, and diseases.

In-depth phenotyping reveals common and novel disease symptoms in a hemizygous knock-in mouse model (Mut-ko/ki) of mut-type methylmalonic aciduria.

Isolated methylmalonic aciduria (MMAuria) is primarily caused by deficiency of methylmalonyl-CoA mutase (MMUT or MUT). Biochemically, MUT deficiency results in the accumulation of methylmalonic acid (MMA), propionyl-carnitine (C3) and other metabolites. Patients often exhibit lethargy, failure to thrive and metabolic decompensation leading to coma or even death, with kidney and neurological impairment frequently identified in the long-term. Here, we report a hemizygous mouse model which combines a knock-in (ki) missense allele of Mut with a knock-out (ko) allele (Mut-ko/ki mice) that was fed a 51%-protein diet from day 12 of life, constituting a bespoke model of MMAuria. Under this diet, mutant mice developed a pronounced metabolic phenotype characterized by drastically increased blood levels of MMA and C3 compared to their littermate controls (Mut-ki/wt). With this bespoke mouse model, we performed a standardized phenotypic screen to assess the whole-body impairments associated with this strong metabolic condition. We found that Mut-ko/ki mice show common clinical manifestations of MMAuria, including pronounced failure to thrive, indications of mild neurological and kidney dysfunction, and degenerative morphological changes in the liver, along with less well described symptoms such as cardiovascular and hematological abnormalities. The analyses also reveal so far unknown disease characteristics, including low bone mineral density, anxiety-related behaviour and ovarian atrophy. This first phenotypic screening of a MMAuria mouse model confirms its relevance to human disease, reveals new alterations associated with MUT deficiency, and suggests a series of quantifiable readouts that can be used to evaluate potential treatment strategies.

Laboratory mouse housing conditions can be improved using common environmental enrichment without compromising data.

Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a "barren" regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.

Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic.

Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.

Systematic phenotyping and correlation of biomarkers with lung function and histology in lung fibrosis.

To date, phenotyping and disease course prediction in idiopathic pulmonary fibrosis (IPF) primarily relies on lung function measures. Blood biomarkers were recently proposed for diagnostic and outcome prediction in IPF, yet their correlation with lung function and histology remains unclear. Here, we comprehensively assessed biomarkers in liquid biopsies and correlated their abundance with lung function and histology during the onset, progression, and resolution of lung fibrosis, with the aim to more precisely evaluate disease progression in the preclinical model of bleomycin-induced pulmonary fibrosis in vivo. Importantly, the strongest correlation of lung function with histological extent of fibrosis was observed at day 14, whereas lung function was unchanged at days 28 and 56, even when histological assessment showed marked fibrotic lesions. Although matrix metalloproteinase-7 (MMP-7), MMP-9, and PAI-1 were significantly elevated in broncheoalveolar lavage of fibrotic mice, only soluble ICAM-1 (sICAM-1) was elevated in the peripheral blood of fibrotic mice and was strongly correlated with the extent of fibrosis. Importantly, tissue-bound ICAM-1 was also elevated in lung homogenates, with prominent staining in hyperplastic type II alveolar epithelial and endothelial cells. In summary, we show that lung function decline is not a prerequisite for histologically evident fibrosis, particularly during the onset or resolution thereof. Plasma levels of sICAM-1 strongly correlate with the extent of lung fibrosis, and may thus be considered for the assessment of intraindividual therapeutic studies in preclinical studies of pulmonary fibrosis.

Temporal and spatial regulation of bone morphogenetic protein signaling in late lung development.

Bone morphogenetic proteins (BMPs) play important roles in early lung development. No study to date has addressed a role for BMP signaling in late lung development. We describe changes in the expression and localization of BMP receptors (Bmpr1a, Bmpr1b, and Bmpr2) and Smad (Smad1, Smad4, Smad5, and Smad8) intracellular signaling proteins during the saccular and alveolarization stages of late lung development. BMP signaling, assessed by Smad1/5 phosphorylation, nuclear translocation, and induction of id1, id2, and id3 gene expression, was evident throughout late lung development. Our data indicate that BMP signaling is active during late lung development, and points to roles for the BMP system in septal and vascular development, and in the homeostasis of the epithelial layer of large conducting airways in the mature lung.

Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia.

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming growth factor (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to 21% or 85% O(2) between postnatal days P1 and P28. Growth and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung fibroblasts cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung fibroblasts to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.