Michelle Stewart

Mouse hitchhiker mutants have spina bifida, dorso-ventral patterning defects and polydactyly: identification of Tulp3 as a novel negative regulator of the Sonic hedgehog pathway.

The mammalian Sonic hedgehog (Shh) signalling pathway is essential for embryonic development and the patterning of multiple organs. Disruption or activation of Shh signalling leads to multiple birth defects, including holoprosencephaly, neural tube defects and polydactyly, and in adults results in tumours of the skin or central nervous system. Genetic approaches with model organisms continue to identify novel components of the pathway, including key molecules that function as positive or negative regulators of Shh signalling. Data presented here define Tulp3 as a novel negative regulator of the Shh pathway. We have identified a new mouse mutant that is a strongly hypomorphic allele of Tulp3 and which exhibits expansion of ventral markers in the caudal spinal cord, as well as neural tube defects and preaxial polydactyly, consistent with increased Shh signalling. We demonstrate that Tulp3 acts genetically downstream of Shh and Smoothened (Smo) in neural tube patterning and exhibits a genetic interaction with Gli3 in limb development. We show that Tulp3 does not appear to alter expression or processing of Gli3, and we demonstrate that transcriptional regulation of other negative regulators (Rab23, Fkbp8, Thm1, Sufu and PKA) is not affected. We discuss the possible mechanism of action of Tulp3 in Shh-mediated signalling in light of these new data.

The mouse Wnt/PCP protein Vangl2 is necessary for migration of facial branchiomotor neurons, and functions independently of Dishevelled

During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration. Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2(Lp/+) embryos did not exacerbate the Vangl2(Lp/+) neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.

Establishing normal plasma and 24-hour urinary biochemistry ranges in C3H, BALB/c and C57BL/6J mice following acclimatization in metabolic cages.

Physiological studies of mice are facilitated by normal plasma and 24-hour urinary reference ranges, but variability of these parameters may increase due to stress that is induced by housing in metabolic cages. We assessed daily weight, food and water intake, urine volume and final day measurements of the following: plasma sodium, potassium, chloride, urea, creatinine, calcium, phosphate, alkaline phosphatase, albumin, cholesterol and glucose; and urinary sodium, potassium, calcium, phosphate, glucose and protein in 24- to 30-week-old C3H/HeH, BALB/cAnNCrl and C57BL/6J mice. Between 15 and 20 mice of each sex from all three strains were individually housed in metabolic cages with ad libitum feeding for up to seven days. Acclimatization was evaluated using general linear modelling for repeated measures and comparison of biochemical data was by unpaired t-test and analysis of variance (SPSS version 12.0.1). Following an initial 5–10% fall in body weight, daily dietary intake, urinary output and weight in all three strains reached stable values after 3–4 days of confinement. Significant differences in plasma glucose, cholesterol, urea, chloride, calcium and albumin, and urinary glucose, sodium, phosphate, calcium and protein were observed between strains and genders. Thus, these results provide normal reference values for plasma and urinary biochemistry in three strains housed in metabolic cages and demonstrate that 3–4 days are required to reach equilibrium in metabolic cage studies. These variations due to strain and gender have significant implications for selecting the appropriate strain upon which to breed genetically-altered models of metabolic and renal disease.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard–Soulier, Bardet–Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

α-Synuclein levels modulate Huntington's disease in mice

α-Synuclein and mutant huntingtin are the major constituents of the intracellular aggregates that characterize the pathology of Parkinsons disease (PD) and Huntingtons disease (HD), respectively. α-Synuclein is likely to be a major contributor to PD, since overexpression of this protein resulting from genetic triplication is sufficient to cause human forms of PD. We have previously demonstrated that wild-type α-synuclein overexpression impairs macroautophagy in mammalian cells and in transgenic mice. Overexpression of human wild-type α-synuclein in cells and Drosophila models of HD worsens the disease phenotype. Here, we examined whether α-synuclein overexpression also worsens the HD phenotype in a mammalian system using two widely used N-terminal HD mouse models (R6/1 and N171-82Q). We also tested the effects of α-synuclein deletion in the same N-terminal HD mouse models, as well as assessed the effects of α-synuclein deletion on macroautophagy in mouse brains. We show that overexpression of wild-type α-synuclein in both mouse models of HD enhances the onset of tremors and has some influence on the rate of weight loss. On the other hand, α-synuclein deletion in both HD models increases autophagosome numbers and this is associated with a delayed onset of tremors and weight loss, two of the most prominent endophenotypes of the HD-like disease in mice. We have therefore established a functional link between these two aggregate-prone proteins in mammals and provide further support for the model that wild-type α-synuclein negatively regulates autophagy even at physiological levels.

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1D83G/D83G mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1D83G/D83G mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1−/−). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.

Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis

Corrochano Sanchez et al. identify a novel mutation (I588V) in SCN4A, which encodes the Nav1.4 voltage-gated sodium channel, in a patient with myotonia and periodic paralysis. By generating and characterizing a mouse model (‘draggen’) carrying the equivalent point mutation (I582V), they uncover novel pathological and metabolic features of SCN4A channelopathies.

Loss-of-Function Mutations in FRRS1L Lead to an Epileptic-Dyskinetic Encephalopathy

Glutamatergic neurotransmission governs excitatory signaling in the mammalian brain, and abnormalities of glutamate signaling have been shown to contribute to both epilepsy and hyperkinetic movement disorders. The etiology of many severe childhood movement disorders and epilepsies remains uncharacterized. We describe a neurological disorder with epilepsy and prominent choreoathetosis caused by biallelic pathogenic variants in FRRS1L, which encodes an AMPA receptor outer-core protein. Loss of FRRS1L function attenuates AMPA-mediated currents, implicating chronic abnormalities of glutamatergic neurotransmission in this monogenic neurological disease of childhood.

Analysing the outcome of CRISPR-aided genome editing in embryos: Screening, genotyping and quality control

The application of CRISPR/Cas9 technology has revolutionised genetics by greatly enhancing the efficacy of genome editing in the early embryo. Furthermore, the system has enabled the generation of allele types previously incompatible with in vivo mutagenesis. Despite its versatility and ease of implementation, CRISPR/Cas9 editing outcome is unpredictable and can generate mosaic founders. Therefore, careful genotyping and characterisation of new mutants is proving essential. The literature presents a wide range of protocols for molecular characterisation, each representing different levels of investment. We present strategies and protocols for designing, producing and screening CRISPR/Cas9 edited founders and genotyping their offspring according to desired allele type (indel, point mutation and deletion).

Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice.

Devoy et al. develop the first mouse model to fully recapitulate human FUS-ALS, as defined by midlife-onset progressive degeneration of motor neurons with dominant inheritance. A toxic gain of function occurs in the absence of FUS protein aggregation, involving disturbance of ribosomes and mitochondria at the endoplasmic reticulum.