[Show abstract][Hide abstract] ABSTRACT: Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.
[Show abstract][Hide abstract] ABSTRACT: Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Patients with WARBM present with a range of clinical symptoms including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely due to the lack of any robust animal models phenocopying both ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18 mutant mouse model of WARBM. Rab18 mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating characteristic ocular features associated with WARBM. In addition, Rab18 mutant cells have an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of WARBM patient cells, as well as cells from patients with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18 mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are most likely not due to gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerve in Rab18 mutant mice reveals significant alterations in several core molecular pathways regulating cytoskeletal dynamics in neurons. The clear similarities between WARBM and the phenotype we describe indicate that the Rab18 mutant mouse provides an important platform for investigating the disease pathogenesis and therapeutic interventions.
Disease Models and Mechanisms 04/2014; · 4.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA) mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna) nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance.
X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver) of FlnaDilp2/+ and wild-type (WT) female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually), consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics.
Mosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.
[Show abstract][Hide abstract] ABSTRACT: Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1(tm1a)) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1(tm1a/tm1a)). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1(tm1a/tm1a) embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.
[Show abstract][Hide abstract] ABSTRACT: Renal-coloboma syndrome, also known as papillorenal syndrome, is an autosomal dominant human disorder in which optic disc coloboma is associated with kidney abnormalities. Mutations in the paired domain transcription factor PAX2 have been found to be the underlying cause of this disease. Disease severity varies between patients, and in some cases, renal hypoplasia has been found in the absence of any retinal defects. Here we report an N-ethyl-N-nitrosourea-induced mouse mutation, Opdc, which is an isoleucinetothreonine missense mutation, I40T, in the first α-helix of the Pax2 paired domain. The mutant protein binds target DNA sequences less strongly than the wild-type protein and acts poorly to transactivate target promoters in culture. The phenotypic consequence of this mutation on the development of the eye and ear is similar to that reported for null alleles of Pax2. However, in homozygotes, cerebellar development is normal on a genetic background in which loss of Pax2 results in failure of cerebellar formation. Moreover, there is a genetic background effect on the heterozygous phenotype such that on some strain backgrounds, kidney development is unaffected. Opdc is the first hypomorphic mutation reported for Pax2 that differs in phenotype from loss-of-function mutations. These results suggest that PAX2 is a strong candidate gene for cases in which human patients have optic disc coloboma not associated with renal dysplasia.
Human Molecular Genetics 10/2010; 20(2):223-34. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear beta-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Pituitary adenylate cyclase activating polypeptide (PACAP) and its high affinity receptor PAC1 are expressed in mammalian retina and involved in processing light information. However, their roles during retinogenesis remain largely elusive. Previously, we have generated transgenic mice overexpressing the human PAC1 receptor, and shown that PACAP signaling is essential for normal development of the central nervous system. In this study, we show for the first time that PACAP signaling plays an important role in the development of retina, particularly in the genesis of GABAergic amacrine cells. Overexpression of the PAC1 receptor leads to an early exit from retinal proliferation, reduced production of GABAergic neurons, and a marked decline in visual function. These data demonstrate that an appropriate level of PACAP signaling is required for normal retinogenesis and visual function. This finding may have implications in GABAergic neuron-related neurological conditions.
[Show abstract][Hide abstract] ABSTRACT: Purpose Characterisation of novel genes involved in vision and eye development from the Harwell EUMODIC screen and a recessive ENU mutagenesis screen.Methods Mice have been generated through the EuComm and EuModic programmes that have knockout mutations in defined genes. In addition, mice with random, undefined, mutations have been produced by chemical mutagenesis. Cohorts of these mice have been subjected to slit lamp examination and indirect ophthalmoscopy, and are tested for visual function using a virtual optokinetic drum with a computer generated image displayed around the test animal. Further phenotypic characterisation involves histology and use of tissue markers. In order to preserve the three-dimensional structure of the eye for examination of the vasculature, for example, we are exploring the use of Optical Projection Tomography which images the intact eye and allows reconstruction of eye structure.Results We have identified a number of mouse lines defective in the visual system. These defects include microphthalmia, eyes open at birth, lens, cornea or iris defects, intraocular bleeding, and retinal degeneration. Furthermore, one line exhibits persistent fetal vasculature in the vitreous (PHPV) which normally regresses after birth through the action of resident macrophages. Determining the basis of this phenotype involves analysis of both the embryonic and adult eye, since mice with defective optic-fissure closure have also been described with this phenotype.Conclusion Characterisation of these mutant lines will enable us to contribute to a more comprehensive understanding of the genetic regulation of eye development and tissue maintenance within the visual system. Many of these mutants are valuable genetic models of human eye diseases.
[Show abstract][Hide abstract] ABSTRACT: The intracellular target of diphtheria toxin is a modified histidine residue, diphthamide, in the translation elongation factor, eEF2 (also known as EFT1). This enigmatic modification occurs in all eukaryotes and is produced in yeast by the action of five gene products, DPH1 to DPH5. Sequence homologues of these genes are present in all sequenced eukaryotic genomes and, in higher eukaryotes, there is functional evidence for DPH1, DPH2, DPH3 and DPH5 acting in diphthamide biosynthesis. We identified a mouse that was mutant for the remaining gene, Dph4. Cells derived from homozygous mutant embryos lacked the diphthamide modification of eEF2 and were resistant to killing by diphtheria toxin. Reporter-tagged DPH4 protein localized to the cytoskeleton, in contrast to the localization of DPH1 and consistent with evidence that DPH4 is not part of a proposed complex containing DPH1, DPH2 and DPH3. Mice that were homozygous for the mutation were retarded in growth and development, and almost always die before birth. Those that survive long enough had preaxial polydactyly, a duplication of digit 1 of the hind foot. This same defect has been seen in embryos that were homozygous for mutation of DPH1, suggesting that lack of diphthamide on eEF2 could result in translational failure of specific proteins, rather than a generalized translation downregulation.
[Show abstract][Hide abstract] ABSTRACT: The melanocortin receptor, MC1R, is a key regulator of pigmentation in mammals, and is necessary for production of dark eumelanin pigment. Human MC1R variants with reduced or absent function are associated with red hair; mouse mutants result in yellow fur. Previous reports indicate differences between mouse and human receptors in their sensitivity to, and requirement for, alphaMSH agonist. We have generated a transgenic mouse model in which coat pigmentation is mediated solely by human MC1R. Although the hair pigment pattern is superficially normal, we show the human receptor is more sensitive to exogenous ligand than mouse Mc1r. Furthermore, although the endogenous receptor antagonist, agouti signalling protein, blocks activation of human MC1R, its action is unlike that on the mouse receptor in that it does not generate an inverse signal. In transfected cells, both receptors show ligand independent signalling. However, in transgenic mice, the human receptor does not elicit significant eumelanin synthesis in absence of ligand, in contrast to the mouse receptor which gives normal eumelanogenesis without ligand. Thus, the mouse model recapitulates the observation that humans mutated in POMC, the melanocortin precursor gene, lack eumelanin and have red hair. We suggest this apparent paradox can be explained by the much lower receptor number expressed in human versus mouse melanocytes, resulting in a much lower endogenous signalling in vivo.
Human Molecular Genetics 11/2007; 16(19):2341-8. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The X-linked gene filamin A (Flna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitrosourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the Flna gene (Y2388X), leading to absence of the Flna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for Flna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.
Human Molecular Genetics 09/2006; 15(16):2457-67. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Members of the type IV collagen family are essential components of all basement membranes (BMs) and define structural stability as well as tissue-specific functions. The major isoform, alpha1.alpha1.alpha2(IV), contributes to the formation of many BMs and its deficiency causes embryonic lethality in mouse. We have identified an allelic series of three ENU induced dominant mouse mutants with missense mutations in the gene Col4a1 encoding the alpha1(IV) subunit chain. Two severe alleles (Bru and Svc) have mutations affecting the conserved glycine residues in the Gly-Xaa-Yaa collagen repeat. Bru heterozygous mice display defects similar to Axenfeld-Rieger anomaly, including iris defects, corneal opacity, vacuolar cataracts, significant iris/corneal adhesions, buphthalmos and optic nerve cupping, a sign indicative of glaucoma. Kidneys of Bru mice have peripheral glomerulopathy characterized by hypertrophy and hyperplasia of the parietal epithelium of Bowman's capsule. A milder allele (Raw) contains a mutation in the Yaa residue of the collagen repeat and was identified by a silvery appearance of the retinal arterioles. All phenotypes are associated with BM defects that affect the eye, kidney and other tissues. This allelic series shows that mutations affecting the collagen domain cause dominant negative effects on the expression and function of the major collagen IV isoform alpha1(IV), and pathological effects vary with the individual mutations.
Human Molecular Genetics 12/2005; 14(21):3161-8. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To identify the underlying molecular defects causing retinal degeneration in seven N-ethyl-N-nitrosourea (ENU) induced mutant alleles of the Pde6b gene and to analyze the timescale of retinal degeneration in these new models of retinitis pigmentosa.
Conformation sensitive capillary electrophoresis and DNA sequencing were used to identify the mutations in the Pde6b gene. Visual acuity testing was performed with a visual-tracking drum at ages ranging from postnatal day 25 to week 10. Retinal examination was performed with an indirect ophthalmoscope. Animals were killed and eyes were prepared for histologic analysis.
Point mutations in the seven new alleles of Pde6b were identified: Three generated premature stop codons, two were missense mutations, and two were splice mutations. The three stop codon mutants and one of the splice mutants had phenotypes indistinguishable from the Pde6b(rd1) mouse in rapidity of onset of retinal degeneration, suggesting that they are null alleles. However, the remaining alleles showed slower onset of retinal degeneration, as determined by visual acuity testing, fundus examination, and histology, indicating that they are hypomorphic alleles.
These data demonstrate a correlation between genotype and phenotype. Four of the mutants with severe genetic lesions have rapid onset of retinal degeneration, as determined by fundus examination. These mice were indistinguishable from Pde6b(rd1) mice, which are effectively blind by 3 weeks of age. In contrast, the milder genetic lesions show a slower progression of the disease and provide the community with models that more closely mimic human retinitis pigmentosa.
[Show abstract][Hide abstract] ABSTRACT: Dilp1 is a semi-dominant mouse mutation that causes dilated pupils when heterozygous and is lethal when homozygous. We report here that it is caused by a point mutation that introduces a stop codon close to the start of the coding sequence of the paired-like homeobox transcription factor Phox2b. Mice carrying a targeted allele of Phox2b also have dilated pupils and the two alleles do not complement. Phox2b is necessary for the development of the autonomic nervous system and when absent one of the consequences is that all parasympathetic ganglia fail to form. Constriction of the pupil is a parasympathetic response mediated by the ciliary ganglion and we find that in Phox2b heterozygous mutants it is highly atrophic. The development of other parasympathetic and sympathetic ganglia appears to be largely unaffected indicating that the ciliary ganglion is exquisitely sensitive to a reduction in dose of this transcription factor. PHOX2B has been implicated in human disease. Mutations, principally leading to polyalanine expansions within the protein, have been found in patients with congenital central hypoventilation syndrome (CCHS), the cardinal feature of which is an inability to breathe unassisted when asleep. Additionally, some CCHS patients have ocular abnormalities, including pupillary defects, although they principally have constricted rather than dilated pupils. The apparent phenotypic differences observed between mice carrying a loss-of-function mutation of Phox2b and CCHS patients indicate that PHOX2B mutations found in CCHS patients, all of which can produce proteins with intact DNA-binding domains, are gain-of-function mutations that alter rather than abolish protein function.
Human Molecular Genetics 08/2004; 13(14):1433-9. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have carried out a genome-wide screen for novel N-ethyl-N-nitrosourea-induced mutations that give rise to eye and vision abnormalities in the mouse and have identified 25 inherited phenotypes that affect all parts of the eye. A combination of genetic mapping, complementation and molecular analysis revealed that 14 of these are mutations in genes previously identified to play a role in eye pathophysiology, namely Pax6, Mitf, Egfr and Pde6b. Many of the others are located in genomic regions lacking candidate genes and these define new loci. Four of the mutants display a similar phenotype of dilated pupils but do not appear to be allelic, and at least two of these are embryonic lethal when homozygous. This collection of eye mutations will be valuable for understanding gene function, for dissecting protein function and as models of human eye disease.
Human Molecular Genetics 05/2002; 11(7):755-67. · 7.69 Impact Factor